Compositions and methods for the therapy and diagnosis of lung cancer

ABSTRACT

Compositions and methods for the therapy and diagnosis of cancer, such as lung cancer, are disclosed. Compositions may comprise one or more lung tumor proteins, immunogenic portions thereof, or polynucleotides that encode such portions. Alternatively, a therapeutic composition may comprise an antigen presenting cell that expresses a lung tumor protein, or a T cell that is specific for cells expressing such a protein. Such compositions may be used, for example, for the prevention and treatment of diseases such as lung cancer. Diagnostic methods based on detecting a lung tumor protein, or mRNA encoding such a protein, in a sample are also provided.

REFERENCE TO RELATED APPLICATIONS

This application is continuation-in-part of U.S. patent application Ser.No. 09/658,824, filed Sep. 8, 2000; U.S. patent application Ser. No.09/651,563, filed Aug. 29, 2000; U.S. patent application Ser. No.09/614,124, filed Jul. 11, 2000; U.S. patent application Ser. No.09/589,184, filed Jun. 5, 2000; U.S. patent application Ser. No.09/560,406, filed Apr. 27, 2000; U.S. patent application Ser. No.09/546,259, filed Apr. 10, 2000; U.S. patent application Ser. No.09/533,077, filed Mar. 22, 2000; U.S. patent application Ser. No.09/519,642, filed Mar. 6, 2000; U.S. patent application Ser. No.09/476,300, filed Dec. 30, 1999; U.S. patent application Ser. No.09/466,867, filed Dec. 17, 1999; U.S. patent application Ser. No.09/419,356, filed Oct. 15, 1999; U.S. patent application Ser. No.09/346,492, filed Jun. 30, 1999; each a CIP of the previous applicationand all pending; and PCT/US00/18061, filed Jun. 30, 1999, pending.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to therapy and diagnosis ofcancer, such as lung cancer. The invention is more specifically relatedto polypeptides comprising at least a portion of a lung tumor protein,and to polynucleotides encoding such polypeptides. Such polypeptides andpolynucleotides may be used in compositions for prevention and treatmentof lung cancer, and for the diagnosis and monitoring of such cancers.

BACKGROUND OF THE INVENTION

Cancer is a significant health problem throughout the world. Althoughadvances have been made in detection and therapy of cancer, no vaccineor other universally successful method for prevention or treatment iscurrently available. Current therapies, which are generally based on acombination of chemotherapy or surgery and radiation, continue to proveinadequate in many patients.

Lung cancer is the primary cause of cancer death among both men andwomen in the U.S., with an estimated 172,000 new cases being reported in1994. The five-year survival rate among all lung cancer patients,regardless of the stage of disease at diagnosis, is only 13%. Thiscontrasts with a five-year survival rate of 46% among cases detectedwhile the disease is still localized. However, only 16% of lung cancersare discovered before the disease has spread.

Early detection is difficult since clinical symptoms are often not seenuntil the disease has reached an advanced stage. Currently, diagnosis isaided by the use of chest x-rays, analysis of the type of cellscontained in sputum and fiberoptic examination of the bronchialpassages. Treatment regimens are determined by the type and stage of thecancer, and include surgery, radiation therapy and/or chemotherapy.

In spite of considerable research into therapies for this and othercancers, lung cancer remains difficult to diagnose and treateffectively. Accordingly, there is a need in the art for improvedmethods for detecting and treating such cancers. The present inventionfulfills these needs and further provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methodsfor the diagnosis and therapy of cancer, such as lung cancer. In oneaspect, the present invention provides polypeptides comprising at leasta portion of a lung tumor protein, or a variant thereof. Certainportions and other variants are immunogenic, such that the ability ofthe variant to react with antigen-specific antisera is not substantiallydiminished. Within certain embodiments, the polypeptide comprises asequence that is encoded by a polynucleotide sequence selected from thegroup consisting of: (a) sequences recited in SEQ ID NO: 1-323, 341-782,784-785, 788, 790, 792, 794, 796, 800-804, 807, 808, 810-826, 878-1664,1668, 1669, 1676, 1680-1805 and 1824; and (c) complements of a sequenceof (a) or (b). In specific embodiments, the polypeptides of the presentinvention comprise at least a portion of a tumor protein that includesan amino acid sequence selected from the group consisting of sequencesrecited in SEQ ID NO: 324-340, 783, 786, 787, 789, 791, 793, 795,797-799, 805, 806, 809, 827, 1667, 1670-1675, 1677-1679, 1806-1822, 1825and variants thereof.

The present invention further provides polynucleotides that encode apolypeptide as described above, or a portion thereof (such as a portionencoding at least 15 amino acid residues of a lung tumor protein),expression vectors comprising such polynucleotides and host cellstransformed or transfected with such expression vectors.

Within other aspects, the present invention provides pharmaceuticalcompositions comprising a polypeptide or polynucleotide as describedabove and a physiologically acceptable carrier.

Within a related aspect of the present invention, vaccines, orimmunogenic compositions, for prophylactic or therapeutic use areprovided. Such vaccines comprise a polypeptide or polynucleotide asdescribed above and an immunostimulant.

The present invention further provides pharmaceutical compositions thatcomprise: (a) an antibody or antigen-binding fragment thereof thatspecifically binds to a lung tumor protein; and (b) a physiologicallyacceptable carrier.

Within further aspects, the present invention provides pharmaceuticalcompositions comprising: (a) an antigen presenting cell that expresses apolypeptide as described above and (b) a pharmaceutically acceptablecarrier or excipient. Antigen presenting cells include dendritic cells,macrophages, monocytes, fibroblasts and B cells.

Within related aspects, vaccines, or immunogenic compositions, areprovided that comprise: (a) an antigen presenting cell that expresses apolypeptide as described above and (b) an immunostimulant.

The present invention further provides, in other aspects, fusionproteins that comprise at least one polypeptide as described above, aswell as polynucleotides encoding such fusion proteins.

Within related aspects, pharmaceutical compositions comprising a fusionprotein, or a polynucleotide encoding a fusion protein, in combinationwith a physiologically acceptable carrier are provided.

Vaccines, or immunogenic compositions, are further provided, withinother aspects, that comprise a fusion protein, or a polynucleotideencoding a fusion protein, in combination with an immunostimulant.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient a pharmaceutical composition or immunogeniccomposition as recited above. The patient may be afflicted with lungcancer, in which case the methods provide treatment for the disease, orpatient considered at risk for such a disease may be treatedprophylactically.

The present invention further provides, within other aspects, methodsfor removing tumor cells from a biological sample, comprising contactinga biological sample with T cells that specifically react with a lungtumor protein, wherein the step of contacting is performed underconditions and for a time sufficient to permit the removal of cellsexpressing the protein from the sample.

Within related aspects, methods are provided for inhibiting thedevelopment of a cancer in a patient, comprising administering to apatient a biological sample treated as described above.

Methods are further provided, within other aspects, for stimulatingand/or expanding T cells specific for a lung tumor protein, comprisingcontacting T cells with one or more of: (i) a polypeptide as describedabove; (ii) a polynucleotide encoding such a polypeptide; and/or (iii)an antigen presenting cell that expresses such a polypeptide; underconditions and for a time sufficient to permit the stimulation and/orexpansion of T cells. Isolated T cell populations comprising T cellsprepared as described above are also provided.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient an effective amount of a T cell population asdescribed above.

The present invention further provides methods for inhibiting thedevelopment of a cancer in a patient, comprising the steps of: (a)incubating CD4⁺ and/or CD8⁺ T cells isolated from a patient with one ormore of: (i) a polypeptide comprising at least an immunogenic portion ofa lung tumor protein; (ii) a polynucleotide encoding such a polypeptide;and (iii) an antigen-presenting cell that expressed such a polypeptide;and (b) administering to the patient an effective amount of theproliferated T cells, and thereby inhibiting the development of a cancerin the patient. Proliferated cells may, but need not, be cloned prior toadministration to the patient.

Within further aspects, the present invention provides methods fordetermining the presence or absence of a cancer in a patient,comprising: (a) contacting a biological sample obtained from a patientwith a binding agent that binds to a polypeptide as recited above; (b)detecting in the sample an amount of polypeptide that binds to thebinding agent; and (c) comparing the amount of polypeptide with apredetermined cut-off value, and therefrom determining the presence orabsence of a cancer in the patient. Within preferred embodiments, thebinding agent is an antibody, more preferably a monoclonal antibody. Thecancer may be lung cancer.

The present invention also provides, within other aspects, methods formonitoring the progression of a cancer in a patient. Such methodscomprise the steps of: (a) contacting a biological sample obtained froma patient at a first point in time with a binding agent that binds to apolypeptide as recited above; (b) detecting in the sample an amount ofpolypeptide that binds to the binding agent; (c) repeating steps (a) and(b) using a biological sample obtained from the patient at a subsequentpoint in time; and (d) comparing the amount of polypeptide detected instep (c) with the amount detected in step (b) and therefrom monitoringthe progression of the cancer in the patient.

The present invention further provides, within other aspects, methodsfor determining the presence or absence of a cancer in a patient,comprising the steps of: (a) contacting a biological sample obtainedfrom a patient, with an oligonucleotide that hybridizes to apolynucleotide that encodes a lung tumor protein; (b) detecting in thesample a level of a polynucleotide, preferably mRNA, that hybridizes tothe oligonucleotide; and (c) comparing the level of polynucleotide thathybridizes to the oligonucleotide with a predetermined cut-off value,and therefrom determining the presence or absence of a cancer in thepatient. Within certain embodiments, the amount of mRNA is detected viapolymerase chain reaction using, for example, at least oneoligonucleotide primer that hybridizes to a polynucleotide encoding apolypeptide as recited above, or a complement of such a polynucleotide.Within other embodiments, the amount of mRNA is detected using ahybridization technique, employing an oligonucleotide probe thathybridizes to a polynucleotide that encodes a polypeptide as recitedabove, or a complement of such a polynucleotide.

In related aspects, methods are provided for monitoring the progressionof a cancer in a patient, comprising the steps of: (a) contacting abiological sample obtained from a patient with an oligonucleotide thathybridizes to a polynucleotide that encodes a lung tumor protein; (b)detecting in the sample an amount of a polynucleotide that hybridizes tothe oligonucleotide; (c) repeating steps (a) and (b) using a biologicalsample obtained from the patient at a subsequent point in time; and (d)comparing the amount of polynucleotide detected in step (c) with theamount detected in step (b) and therefrom monitoring the progression ofthe cancer in the patient.

Within further aspects, the present invention provides antibodies, suchas monoclonal antibodies, that bind to a polypeptide as described above,as well as diagnostic kits comprising such antibodies. Diagnostic kitscomprising one or more oligonucleotide probes or primers as describedabove are also provided.

These and other aspects of the present invention will become apparentupon reference to the following detailed description. All referencesdisclosed herein are hereby incorporated by reference in their entiretyas if each was incorporated individually.

SEQUENCE IDENTIFIERS

SEQ ID NO: 1 is the determined cDNA sequence for clone #19038, alsoreferred to as L845P.

SEQ ID NO: 2 is the determined cDNA sequence for clone #19036.

SEQ ID NO: 3 is the determined cDNA sequence for clone #19034.

SEQ ID NO: 4 is the determined cDNA sequence for clone #19033.

SEQ ID NO: 5 is the determined cDNA sequence for clone #19032.

SEQ ID NO: 6 is the determined cDNA sequence for clone #19030, alsoreferred to as L559S.

SEQ ID NO: 7 is the determined cDNA sequence for clone #19029.

SEQ ID NO: 8 is the determined cDNA sequence for clone #19025.

SEQ ID NO: 9 is the determined cDNA sequence for clone #19023.

SEQ ID NO: 10 is the determined cDNA sequence for clone #18929.

SEQ ID NO: 11 is the determined cDNA sequence for clone #19010.

SEQ ID NO: 12 is the determined cDNA sequence for clone #19009.

SEQ ID NO: 13 is the determined cDNA sequence for clones #19005, 19007,19016 and 19017.

SEQ ID NO: 14 is the determined cDNA sequence for clone #19004.

SEQ ID NO: 15 is the determined cDNA sequence for clones #19002 and18965.

SEQ ID NO: 16 is the determined cDNA sequence for clone #18998.

SEQ ID NO: 17 is the determined cDNA sequence for clone #18997.

SEQ ID NO: 18 is the determined cDNA sequence for clone #18996.

SEQ ID NO: 19 is the determined cDNA sequence for clone #18995.

SEQ ID NO: 20 is the determined cDNA sequence for clone #18994, alsoknown as L846P.

SEQ ID NO: 21 is the determined cDNA sequence for clone #18992.

SEQ ID NO: 22 is the determined cDNA sequence for clone #18991.

SEQ ID NO: 23 is the determined cDNA sequence for clone #18990, alsoreferred to as clone #20111.

SEQ ID NO: 24 is the determined cDNA sequence for clone #18987.

SEQ ID NO: 25 is the determined cDNA sequence for clone #18985, alsoreferred as L839P.

SEQ ID NO: 26 is the determined cDNA sequence for clone #18984, alsoreferred to as L847P.

SEQ ID NO: 27 is the determined cDNA sequence for clone #18983.

SEQ ID NO: 28 is the determined cDNA sequence for clones #18976 and18980.

SEQ ID NO: 29 is the determined cDNA sequence for clone #18975.

SEQ ID NO: 30 is the determined cDNA sequence for clone #18974.

SEQ ID NO: 31 is the determined cDNA sequence for clone #18973.

SEQ ID NO: 32 is the determined cDNA sequence for clone #18972.

SEQ ID NO: 33 is the determined cDNA sequence for clone #18971, alsoreferred to as L801P.

SEQ ID NO: 34 is the determined cDNA sequence for clone #18970.

SEQ ID NO: 35 is the determined cDNA sequence for clone #18966.

SEQ ID NO: 36 is the determined cDNA sequence for clones #18964, 18968and 19039.

SEQ ID NO: 37 is the determined cDNA sequence for clone #18960.

SEQ ID NO: 38 is the determined cDNA sequence for clone #18959.

SEQ ID NO: 39 is the determined cDNA sequence for clones #18958 and18982.

SEQ ID NO: 40 is the determined cDNA sequence for clones #18956 and19015.

SEQ ID NO: 41 is the determined cDNA sequence for clone #18954, alsoreferred to L848P.

SEQ ID NO: 42 is the determined cDNA sequence for clone #18951.

SEQ ID NO: 43 is the determined cDNA sequence for clone #18950.

SEQ ID NO: 44 is the determined cDNA sequence for clones #18949 and19024, also referred to as L844P.

SEQ ID NO: 45 is the determined cDNA sequence for clone #18948.

SEQ ID NO: 46 is the determined cDNA sequence for clone #18947, alsoreferred to as L840P.

SEQ ID NO: 47 is the determined cDNA sequence for clones #18946, 18953,18969 and 19027.

SEQ ID NO: 48 is the determined cDNA sequence for clone #18942.

SEQ ID NO: 49 is the determined cDNA sequence for clone #18940, 18962,18963, 19006, 19008, 19000, and 19031.

SEQ ID NO: 50 is the determined cDNA sequence for clone #18939.

SEQ ID NO: 51 is the determined cDNA sequence for clones #18938 and18952.

SEQ ID NO: 52 is the determined cDNA sequence for clone #18938.

SEQ ID NO: 53 is the determined cDNA sequence for clone #18937.

SEQ ID NO: 54 is the determined cDNA sequence for clones #18934, 18935,18993 and 19022, also referred to as L548S.

SEQ ID NO: 55 is the determined CDNA sequence for clone #18932.

SEQ ID NO: 56 is the determined cDNA sequence for clones #18931 and18936.

SEQ ID NO: 57 is the determined cDNA sequence for clone #18930.

SEQ ID NO: 58 is the determined CDNA sequence for clone #19014, alsoreferred to as L773P.

SEQ ID NO: 59 is the determined CDNA sequence for clone #19127.

SEQ ID NO: 60 is the determined CDNA sequence for clones #19057 and19064.

SEQ ID NO: 61 is the determined cDNA sequence for clone #19122.

SEQ ID NO: 62 is the determined cDNA sequence for clones #19120 and18121.

SEQ ID NO: 63 is the determined CDNA sequence for clone #19118.

SEQ ID NO: 64 is the determined CDNA sequence for clone #19117.

SEQ ID NO: 65 is the determined cDNA sequence for clone #19116.

SEQ ID NO: 66 is the determined cDNA sequence for clone #19114.

SEQ ID NO: 67 is the determined CDNA sequence for clone #19112, alsoknown as L561S.

SEQ ID NO: 68 is the determined cDNA sequence for clone #19110.

SEQ ID NO: 69 is the determined cDNA sequence for clone #19107, alsoreferred to as L552S.

SEQ ID NO: 70 is the determined cDNA sequence for clone #19106, alsoreferred to as L547S.

SEQ ID NO: 71 is the determined cDNA sequence for clones #19105 and19111.

SEQ ID NO: 72 is the determined CDNA sequence for clone #19099.

SEQ ID NO: 73 is the determined cDNA sequence for clones #19095, 19104and 19125, also referred to as L549S.

SEQ ID NO: 74 is the determined cDNA sequence for clone #19094.

SEQ ID NO: 75 is the determined cDNA sequence for clones #19089 and19101.

SEQ ID NO: 76 is the determined cDNA sequence for clone #19088.

SEQ ID NO: 77 is the determined cDNA sequence for clones #19087, 19092,19096, 19100 and 19119.

SEQ ID NO: 78 is the determined cDNA sequence for clone #19086.

SEQ ID NO: 79 is the determined CDNA sequence for clone #19085, alsoreferred to as L550S.

SEQ ID NO: 80 is the determined cDNA sequence for clone #19084, alsoreferred to as clone #19079.

SEQ ID NO: 81 is the determined cDNA sequence for clone #19082.

SEQ ID NO: 82 is the determined CDNA sequence for clone #19080.

SEQ ID NO: 83 is the determined CDNA sequence for clone #19077.

SEQ ID NO: 84 is the determined CDNA sequence for clone #19076, alsoreferred to as L551S.

SEQ ID NO: 85 is the determined cDNA sequence for clone #19074, alsoreferred to as clone #20102.

SEQ ID NO: 86 is the determined cDNA sequence for clone #19073, alsoreferred to as L560S.

SEQ ID NO: 87 is the determined cDNA sequence for clones #19072 and19115.

SEQ ID NO: 88 is the determined cDNA sequence for clone #19071.

SEQ ID NO: 89 is the determined cDNA sequence for clone #19070.

SEQ ID NO: 90 is the determined cDNA sequence for clone #19069.

SEQ ID NO: 91 is the determined cDNA sequence for clone #19068, alsoreferred to L563S.

SEQ ID NO: 92 is the determined cDNA sequence for clone #19066.

SEQ ID NO: 93 is the determined cDNA sequence for lone #19065.

SEQ ID NO: 94 is the determined cDNA sequence for clone #19063.

SEQ ID NO: 95 is the determined cDNA sequence for clones #19061, 19081,19108 and 19109.

SEQ ID NO: 96 is the determined cDNA sequence for clones #19060, 19067and 19083, also referred to as L548S.

SEQ ID NO: 97 is the determined cDNA sequence for clones #19059 and19062.

SEQ ID NO: 98 is the determined CDNA sequence for clone #19058.

SEQ ID NO: 99 is the determined cDNA sequence for clone #19124.

SEQ ID NO: 100 is the determined cDNA sequence for clone #18929.

SEQ ID NO: 101 is the determined cDNA sequence for clone #18422.

SEQ ID NO: 102 is the determined cDNA sequence for clone #18425.

SEQ ID NO: 103 is the determined cDNA sequence for clone #18431.

SEQ ID NO: 104 is the determined CDNA sequence for clone #18433.

SEQ ID NO: 105 is the determined cDNA sequence for clone #18444.

SEQ ID NO: 106 is the determined cDNA sequence for clone #18449.

SEQ ID NO: 107 is the determined cDNA sequence for clone #18451.

SEQ ID NO: 108 is the determined cDNA sequence for clone #18452.

SEQ ID NO: 109 is the determined cDNA sequence for clone #18455.

SEQ ID NO: 110 is the determined CDNA sequence for clone #18457.

SEQ ID NO: 111 is the determined cDNA sequence for clone #18466.

SEQ ID NO: 112 is the determined cDNA sequence for clone #18468.

SEQ ID NO: 113 is the determined cDNA sequence for clone #18471.

SEQ ID NO: 114 is the determined cDNA sequence for clone #18475.

SEQ ID NO: 115 is the determined cDNA sequence for clone #18476.

SEQ ID NO: 116 is the determined cDNA sequence for clone #18477.

SEQ ID NO: 117 is the determined cDNA sequence for clone #20631.

SEQ ID NO: 118 is the determined cDNA sequence for clone #20634.

SEQ ID NO: 119 is the determined cDNA sequence for clone #20635.

SEQ ID NO: 120 is the determined cDNA sequence for clone #20637.

SEQ ID NO: 121 is the determined cDNA sequence for clone #20638.

SEQ ID NO: 122 is the determined cDNA sequence for clone #20643.

SEQ ID NO: 123 is the determined cDNA sequence for clone #20652.

SEQ ID NO: 124 is the determined cDNA sequence for clone #20653.

SEQ ID NO: 125 is the determined cDNA sequence for clone #20657.

SEQ ID NO: 126 is the determined cDNA sequence for clone #20658.

SEQ ID NO: 127 is the determined cDNA sequence for clone #20660.

SEQ ID NO: 128 is the determined cDNA sequence for clone #20661.

SEQ ID NO: 129 is the determined cDNA sequence for clone #20663.

SEQ ID NO: 130 is the determined cDNA sequence for clone #20665.

SEQ ID NO: 132 is the determined cDNA sequence for clone #20670.

SEQ ID NO: 132 is the determined cDNA sequence for clone #20671.

SEQ ID NO: 133 is the determined cDNA sequence for clone #20672.

SEQ ID NO: 134 is the determined cDNA sequence for clone #20675.

SEQ ID NO: 135 is the determined CDNA sequence for clone #20679.

SEQ ID NO: 136 is the determined cDNA sequence for clone #20681.

SEQ ID NO: 137 is the determined cDNA sequence for clone #20682.

SEQ ID NO: 138 is the determined cDNA sequence for clone #20684.

SEQ ID NO: 139 is the determined cDNA sequence for clone #20685.

SEQ ID NO: 140 is the determined cDNA sequence for clone #20689.

SEQ ID NO: 141 is the determined cDNA sequence for clone #20699.

SEQ ID NO: 142 is the determined cDNA sequence for clone #20701.

SEQ ID NO: 143 is the determined cDNA sequence for clone #20702.

SEQ ID NO: 144 is the determined cDNA sequence for clone #20708.

SEQ ID NO: 145 is the determined cDNA sequence for clone #20715.

SEQ ID NO: 146 is the determined cDNA sequence for clone #20716.

SEQ ID NO: 147 is the determined cDNA sequence for clone #20719.

SEQ ID NO: 148 is the determined cDNA sequence for clone #19129.

SEQ ID NO: 149 is the determined cDNA sequence for clone #19131.1.

SEQ ID NO: 150 is the determined cDNA sequence for clone #19132.2.

SEQ ID NO: 151 is the determined cDNA sequence for clone #19133.

SEQ ID NO: 152 is the determined cDNA sequence for clone #19134.2.

SEQ ID NO: 153 is the determined cDNA sequence for clone #19135.2.

SEQ ID NO: 154 is the determined cDNA sequence for clone #19137.

SEQ ID NO: 155 is a first determined cDNA sequence for clone #19138.1.

SEQ ID NO: 156 is a second determined cDNA sequence for clone #19138.2.

SEQ ID NO: 157 is the determined cDNA sequence for clone #19139.

SEQ ID NO: 158 is a first determined cDNA sequence for clone #19140.1.

SEQ ID NO: 159 is a second determined cDNA sequence for clone #19140.2.

SEQ ID NO: 160 is the determined cDNA sequence for clone #19141.

SEQ ID NO: 161 is the determined cDNA sequence for clone #19143.

SEQ ID NO: 162 is the determined cDNA sequence for clone #19144.

SEQ ID NO: 163 is a first determined cDNA sequence for clone #19145.1.

SEQ ID NO: 164 is a second determined cDNA sequence for clone 145.2.

SEQ ID NO: 165 is the determined cDNA sequence for clone #19146.

SEQ ID NO: 166 is the determined cDNA sequence for clone # 19149.1.

SEQ ID NO: 167 is the determined cDNA sequence for clone #19152.

SEQ ID NO: 168 is a first determined cDNA sequence for clone #19153.1.

SEQ ID NO: 169 is a second determined cDNA sequence for clone 19153.2.

SEQ ID NO: 170 is the determined cDNA sequence for clone #19155.

SEQ ID NO: 171 is the determined cDNA sequence for clone #19157.

SEQ ID NO: 172 is the determined cDNA sequence for clone #19159.

SEQ ID NO: 173 is the determined cDNA sequence for clone 19160.

SEQ ID NO: 174 is a first determined cDNA sequence for clone #19161.1.

SEQ ID NO: 175 is a second determined cDNA sequence for clone #19161.2.

SEQ ID NO: 176 is the determined cDNA sequence for clone #19162.1.

SEQ ID NO: 177 is the determined cDNA sequence for clone #19166.

SEQ ID NO: 178 is the determined cDNA sequence for clone #19169.

SEQ ID NO: 179 is the determined cDNA sequence for clone #19171.

SEQ ID NO: 180 is a first determined cDNA sequence for clone #19173.1.

SEQ ID NO: 181 is a second determined cDNA sequence for clone #19173.2.

SEQ ID NO: 182 is the determined cDNA sequence for clone #19174.1.

SEQ ID NO: 183 is the determined cDNA sequence for clone #19175.

SEQ ID NO: 184 is the determined cDNA sequence for clone #19177.

SEQ ID NO: 185 is the determined cDNA sequence for clone #19178.

SEQ ID NO: 186 is the determined cDNA sequence for clone #19179.1.

SEQ ID NO: 187 is the determined cDNA sequence for clone #19179.2.

SEQ ID NO: 188 is the determined cDNA sequence for clone #19180.

SEQ ID NO: 189 is a first determined cDNA sequence for clone #19182.1.

SEQ ID NO: 190 is a second determined cDNA sequence for clone 19182.2.

SEQ ID NO: 191 is the determined cDNA sequence for clone #19183.1.

SEQ ID NO: 192 is the determined cDNA sequence for clone #19185.1.

SEQ ID NO: 193 is the determined cDNA sequence for clone #19187.

SEQ ID NO: 194 is the determined cDNA sequence for clone #19188.

SEQ ID NO: 195 is the determined cDNA sequence for clone #19190.

SEQ ID NO: 196 is the determined cDNA sequence for clone #19191.

SEQ ID NO: 197 is the determined cDNA sequence for clone #19192.

SEQ ID NO: 198 is the determined cDNA sequence for clone #19193.

SEQ ID NO: 199 is a first determined cDNA sequence for clone #19194. 1.

SEQ ID NO: 200 is a second determined cDNA sequence for clone #19194.2.

SEQ ID NO: 201 is the determined cDNA sequence for clone #19197.

SEQ ID NO: 202 is a first determined cDNA sequence for clone #19200.1.

SEQ ID NO: 203 is a second determined cDNA sequence for clone #19200.2.

SEQ ID NO: 204 is the determined cDNA sequence for clone #19202.

SEQ ID NO: 205 is a first determined cDNA sequence for clone #19204.1.

SEQ ID NO: 206 is a second determined cDNA sequence for clone #19204.2.

SEQ ID NO: 207 is the determined cDNA sequence for clone #19205.

SEQ ID NO: 208 is a first determined cDNA sequence for clone #19206.1.

SEQ ID NO: 209 is a second determined cDNA sequence for clone #19206.2.

SEQ ID NO: 210 is the determined cDNA sequence for clone #19207.

SEQ ID NO: 211 is the determined cDNA sequence for clone #19208.

SEQ ID NO: 212 is a first determined cDNA sequence for clone #19211.1.

SEQ ID NO: 213 is a second deter mined cDNA sequence for clone #19211.2.

SEQ ID NO: 214 is a first determined cDNA sequence for clone #19214.1.

SEQ ID NO: 215 is a second determined cDNA sequence for clone #19214.2.

SEQ ID NO: 216 is the determined CDNA sequence for clone #19215.

SEQ ID NO: 217 is a first determined cDNA sequence for clone #19217. 2.

SEQ ID NO: 218 is a second determined cDNA sequence for clone #19217.2.

SEQ ID NO: 219 is a first determined cDNA sequence for clone #19218.1.

SEQ ID NO: 220 is a second determined cDNA sequence for clone #19218.2.

SEQ ID NO: 221 is a first determined CDNA sequence for cone #19220.1.

SEQ ID NO: 222 is a second determined cDNA sequence for clone #19220.2.

SEQ ID NO: 223 is the determined CDNA sequence for clone #22015.

SEQ ID NO: 224 is the determined cDNA sequence for clone #22017.

SEQ ID NO: 225 is the determined cDNA sequence for clone #22019.

SEQ ID NO: 226 is the determined cDNA sequence for clone #22020.

SEQ ID NO: 227 is the determined cDNA sequence for clone #22023.

SEQ ID NO: 228 is the determined CDNA sequence for clone #22026.

SEQ ID NO: 229 is the determined CDNA sequence for clone #22027.

SEQ ID NO: 230 is the determined CDNA sequence for clone #22028.

SEQ ID NO: 231 is the determined cDNA sequence for clone #22032.

SEQ ID NO: 232 is the determined cDNA sequence for clone #22037.

SEQ ID NO: 233 is the determined cDNA sequence for clone #22045.

SEQ ID NO: 234 is the determined cDNA sequence for clone #22048.

SEQ ID NO: 235 is the determined cDNA sequence for clone #22050.

SEQ ID NO: 236 is the determined cDNA sequence for clone #22052.

SEQ ID NO: 237 is the determined cDNA sequence for clone #22053.

SEQ ID NO: 238 is the determined cDNA sequence for clone #22057.

SEQ ID NO: 239 is the determined cDNA sequence for clone #22066.

SEQ ID NO: 240 is the determined cDNA sequence for clone #22077.

SEQ ID NO: 241 is the determined cDNA sequence for clone #22085.

SEQ ID NO: 242 is the determined cDNA sequence for clone #22105.

SEQ ID NO: 243 is the determined cDNA sequence for clone #22108.

SEQ ID NO: 244 is the determined cDNA sequence for clone #22109.

SEQ ID NO: 245 is the determined cDNA sequence for clone #24842.

SEQ ID NO: 246 is the determined cDNA sequence for clone #24843.

SEQ ID NO: 247 is the determined cDNA sequence for clone #24845.

SEQ ID NO: 248 is the determined cDNA sequence for clone #24851.

SEQ ID NO: 249 is the determined cDNA sequence for clone #24852.

SEQ ID NO: 250 is the determined cDNA sequence for clone #24853.

SEQ ID NO: 251 is the determined cDNA sequence for clone #24854.

SEQ ID NO: 252 is the determined cDNA sequence for clone #24855.

SEQ ID NO: 253 is the determined cDNA sequence for clone #24860.

SEQ ID NO: 254 is the determined cDNA sequence for clone #24864.

SEQ ID NO: 255 is the determined cDNA sequence for clone #24866.

SEQ ID NO: 256 is the determined cDNA sequence for clone #24867.

SEQ ID NO: 257 is the determined cDNA sequence for clone #24868.

SEQ ID NO: 258 is the determined cDNA sequence for clone #24869.

SEQ ID NO: 259 is the determined cDNA sequence for clone #24870.

SEQ ID NO: 260 is the determined cDNA sequence for clone #24872.

SEQ ID NO: 261 is the determined cDNA sequence for clone #24873.

SEQ ID NO: 262 is the determined cDNA sequence for clone #24875.

SEQ ID NO: 263 is the determined cDNA sequence for clone #24882.

SEQ ID NO: 264 is the determined cDNA sequence for clone #24885.

SEQ ID NO: 265 is the determined cDNA sequence for clone #24886.

SEQ ID NO: 266 is the determined cDNA sequence for clone #24887.

SEQ ID NO: 267 is the determined cDNA sequence for clone #24888.

SEQ ID NO: 268 is the determined cDNA sequence for clone #24890.

SEQ ID NO: 269 is the determined cDNA sequence for clone #24896.

SEQ ID NO: 270 is the determined cDNA sequence for clone #24897.

SEQ ID NO: 271 is the determined cDNA sequence for clone #24899.

SEQ ID NO: 272 is the determined cDNA sequence for clone #24901.

SEQ ID NO: 273 is the determined cDNA sequence for clone #24902.

SEQ ID NO: 274 is the determined cDNA sequence for clone #24906.

SEQ ID NO: 275 is the determined cDNA sequence for clone #24912.

SEQ ID NO: 276 is the determined cDNA sequence for clone #24913.

SEQ ID NO: 277 is the determined cDNA sequence for clone #24920.

SEQ ID NO: 278 is the determined cDNA sequence for clone #24927.

SEQ ID NO: 279 is the determined cDNA sequence for clone #24930.

SEQ ID NO: 280 is the determined cDNA sequence for clone #26938.

SEQ ID NO: 281 is the determined cDNA sequence for clone #26939.

SEQ ID NO: 282 is the determined cDNA sequence for clone #26943.

SEQ ID NO: 283 is the determined cDNA sequence for clone #26948.

SEQ ID NO: 284 is the determined cDNA sequence for clone #26951.

SEQ ID NO: 285 is the determined cDNA sequence for clone #26955.

SEQ ID NO: 286 is the determined cDNA sequence for clone #26956.

SEQ ID NO: 287 is the determined cDNA sequence for clone #26959.

SEQ ID NO: 288 is the determined cDNA sequence for clone #26961.

SEQ ID NO: 289 is the determined cDNA sequence for clone #26962.

SEQ ID NO: 290 is the determined cDNA sequence for clone #26964.

SEQ ID NO: 291 is the determined cDNA sequence for clone #26966.

SEQ ID NO: 292 is the determined cDNA sequence for clone #26968.

SEQ ID NO: 293 is the determined cDNA sequence for clone #26972.

SEQ ID NO: 294 is the determined cDNA sequence for clone #26973.

SEQ ID NO: 295 is the determined cDNA sequence for clone #26974.

SEQ ID NO: 296 is the determined cDNA sequence for clone #26976.

SEQ ID NO: 297 is the determined cDNA sequence for clone #26977.

SEQ ID NO: 298 is the determined cDNA sequence for clone #26979.

SEQ ID NO: 299 is the determined cDNA sequence for clone #26980.

SEQ ID NO: 300 is the determined cDNA sequence for clone #26981.

SEQ ID NO: 301 is the determined cDNA sequence for clone #26984.

SEQ ID NO: 302 is the determined cDNA sequence for clone #26985.

SEQ ID NO: 303 is the determined cDNA sequence for clone #26986.

SEQ ID NO: 304 is the determined cDNA sequence for clone #26993.

SEQ ID NO: 305 is the determined cDNA sequence for clone #26994.

SEQ ID NO: 306 is the determined cDNA sequence for clone #26995.

SEQ ID NO: 307 is the determined cDNA sequence for clone #27003.

SEQ ID NO: 308 is the determined cDNA sequence for clone #27005.

SEQ ID NO: 309 is the determined cDNA sequence for clone #27010.

SEQ ID NO: 310 is the determined cDNA sequence for clone #27011.

SEQ ID NO: 311 is the determined cDNA sequence for clone #27013.

SEQ ID NO: 312 is the determined cDNA sequence for clone #27016

SEQ ID NO: 313 is the determined cDNA sequence for clone #27017.

SEQ ID NO: 314 is the determined cDNA sequence for clone #27019.

SEQ ID NO: 315 is the determined cDNA sequence for clone #27028.

SEQ ID NO: 316 is the full-length cDNA sequence for clone #19060.

SEQ ID NO: 317 is the full-length cDNA sequence for clone #18964.

SEQ ID NO: 318 is the fell-length cDNA sequence for clone #18929.

SEQ ID NO: 319 is the full-length cDNA sequence for clone #18991.

SEQ ID NO: 320 is the full-length cDNA sequence for clone #18996.

SEQ ID NO: 321 is the full-length cDNA sequence for clone #18966.

SEQ ID NO: 322 is the full-length cDNA sequence for clone #18951.

SEQ ID NO: 323 is the full-length cDNA sequence for clone #18973 (alsoknown as L516S).

SEQ ID NO: 324 is the amino acid sequence for clone #19060.

SEQ ID NO: 325 is the amino acid sequence for clone #19063.

SEQ ID NO: 326 is the amino acid sequence for clone #19077.

SEQ ID NO: 327 is the amino acid sequence for clone #19110.

SEQ ID NO: 328 is the amino acid sequence for clone #19122.

SEQ ID NO: 329 is the amino acid sequence for clone #19118.

SEQ ID NO: 330 is the amino acid sequence for clone #19080.

SEQ ID NO: 331 is the amino acid sequence for clone #19127.

SEQ ID NO: 332 is the amino acid sequence for clone #19117.

SEQ ID NO: 333 is the amino acid sequence for clone #19095, alsoreferred to L549S.

SEQ ID NO: 334 is the amino acid sequence for clone #18964.

SEQ ID NO: 335 is the amino acid sequence for clone #18929.

SEQ ID NO: 336 is the amino acid sequence for clone #18991.

SEQ ID NO: 337 is the amino acid sequence for clone #18996.

SEQ ID NO: 338 is the amino acid sequence for clone #18966.

SEQ ID NO: 339 is the amino acid sequence for clone #18951.

SEQ ID NO: 340 is the amino acid sequence for clone #18973.

SEQ ID NO: 341 is the determined cDNA sequence for clone 26461.

SEQ ID NO: 342 is the determined cDNA sequence for clone 26462.

SEQ ID NO: 343 is the determined cDNA sequence for clone 26463.

SEQ ID NO: 344 is the determined cDNA sequence for clone 26464.

SEQ ID NO: 345 is the determined cDNA sequence for clone 26465.

SEQ ID NO: 346 is the determined cDNA sequence for clone 26466.

SEQ ID NO: 347 is the determined cDNA sequence for clone 26467.

SEQ ID NO: 348 is the determined cDNA sequence for clone 26468.

SEQ ID NO: 349 is the determined cDNA sequence for clone 26469.

SEQ ID NO: 350 is the determined CDNA sequence for clone 26470.

SEQ ID NO: 351 is the determined cDNA sequence for clone 26471.

SEQ ID NO: 352 is the determined cDNA sequence for clone 26472.

SEQ ID NO: 353 is the determined cDNA sequence for clone 26474.

SEQ ID NO: 354 is the determined cDNA sequence for clone 26475.

SEQ ID NO: 355 is the determined cDNA sequence for clone 26476.

SEQ ID NO: 356 is the determined cDNA sequence for clone 26477.

SEQ ID NO: 357 is the determined cDNA sequence for clone 26478.

SEQ ID NO: 358 is the determined cDNA sequence for clone 26479.

SEQ ID NO: 359 is the determined cDNA sequence for clone 26480.

SEQ ID NO: 360 is the determined cDNA sequence for clone 26481.

SEQ ID NO: 361 is the determined cDNA sequence for clone 26482

SEQ ID NO: 362 is the determined cDNA sequence for clone 26483.

SEQ ID NO: 363 is the determined cDNA sequence for clone 26484.

SEQ ID NO: 364 is the determined cDNA sequence for clone 26485.

SEQ ID NO: 365 is the determined cDNA sequence for clone 26486.

SEQ ID NO: 366 is the determined cDNA sequence for clone 26487.

SEQ ID NO: 367 is the determined cDNA sequence for clone 26488.

SEQ ID NO: 368 is the determined cDNA sequence for clone 26489.

SEQ ID NO: 369 is the determined cDNA sequence for clone 26490.

SEQ ID NO: 370 is the determined cDNA sequence for clone 26491.

SEQ ID NO: 371 is the determined cDNA sequence for clone 26492.

SEQ ID NO: 372 is the determined cDNA sequence for clone 26493.

SEQ ID NO: 373 is the determined cDNA sequence for clone 26494.

SEQ ID NO: 374 is the determined cDNA sequence for clone 26495.

SEQ ID NO: 375 is the determined cDNA sequence for clone 26496.

SEQ ID NO: 376 is the determined cDNA sequence for clone 26497.

SEQ ID NO: 377 is the determined cDNA sequence for clone 26498.

SEQ ID NO: 378 is the determined cDNA sequence for clone 26499.

SEQ ID NO: 379 is the determined cDNA sequence for clone 26500.

SEQ ID NO: 380 is the determined cDNA sequence for clone 26501.

SEQ ID NO: 381 is the determined CDNA sequence for clone 26502.

SEQ ID NO: 382 is the determined cDNA sequence for clone 26503.

SEQ ID NO: 383 is the determined cDNA sequence for clone 26504.

SEQ ID NO: 384 is the determined cDNA sequence for clone 26505.

SEQ ID NO: 385 is the determined cDNA sequence for clone 26506.

SEQ ID NO: 386 is the determined cDNA sequence for clone 26507.

SEQ ID NO: 387 is the determined cDNA sequence for clone 26508.

SEQ ID NO: 388 is the determined cDNA sequence for clone 26509.

SEQ ID NO: 389 is the determined cDNA sequence for clone 26511.

SEQ ID NO: 390 is the determined cDNA sequence for clone 26513.

SEQ ID NO: 391 is the determined cDNA sequence for clone 26514.

SEQ ID NO: 392 is the determined cDNA sequence for clone 26515.

SEQ ID NO: 393 is the determined cDNA sequence for clone 26516.

SEQ ID NO: 394 is the determined cDNA sequence for clone 26517.

SEQ ID NO: 395 is the determined cDNA sequence for clone 26518.

SEQ ID NO: 396 is the determined cDNA sequence for clone 26519.

SEQ ID NO: 397 is the determined cDNA sequence for clone 26520.

SEQ ID NO: 398 is the determined cDNA sequence for clone 26521.

SEQ ID NO: 399 is the determined cDNA sequence for clone 26522.

SEQ ID NO: 400 is the determined cDNA sequence for clone 26523.

SEQ ID NO: 401 is the determined cDNA sequence for clone 26524.

SEQ ID NO: 402 is the determined cDNA sequence for clone 26526.

SEQ ID NO: 403 is the determined cDNA sequence for clone 26527.

SEQ ID NO: 404 is the determined cDNA sequence for clone 26528.

SEQ ID NO: 405 is the determined cDNA sequence for clone 26529.

SEQ ID NO: 406 is the determined cDNA sequence for clone 26530.

SEQ ID NO: 407 is the determined cDNA sequence for clone 26532.

SEQ ID NO: 408 is the determined CDNA sequence for clone 26533.

SEQ ID NO: 409 is the determined cDNA sequence for clone 26534.

SEQ ID NO: 410 is the determined cDNA sequence for clone 26535.

SEQ ID NO: 411 is the determined cDNA sequence for clone 26536.

SEQ ID NO: 412 is the determined cDNA sequence for clone 26537.

SEQ ID NO: 413 is the determined cDNA sequence for clone 26538.

SEQ ID NO: 414 is the determined cDNA sequence for clone 26540.

SEQ ID NO: 415 is the determined cDNA sequence for clone 26541.

SEQ ID NO: 416 is the determined cDNA sequence for clone 26542.

SEQ ID NO: 417 is the determined cDNA sequence for clone 26543.

SEQ ID NO: 418 is the determined cDNA sequence for clone 26544.

SEQ ID NO: 419 is the determined cDNA sequence for clone 26546.

SEQ ID NO: 420 is the determined cDNA sequence for clone 26547.

SEQ ID NO: 421 is the determined cDNA sequence for clone 26548.

SEQ ID NO: 422 is the determined cDNA sequence for clone 26549.

SEQ ID NO: 423 is the determined cDNA sequence for clone 26550.

SEQ ID NO: 424 is the determined cDNA sequence for clone 26551.

SEQ ID NO: 425 is the determined cDNA sequence for clone 26552.

SEQ ID NO: 426 is the determined cDNA sequence for clone 26553.

SEQ ID NO: 427 is the determined cDNA sequence for clone 26554.

SEQ ID NO: 428 is the determined cDNA sequence for clone 26556.

SEQ ID NO: 429 is the determined cDNA sequence for clone 26557.

SEQ ID NO: 430 is the determined cDNA sequence for clone 27631.

SEQ ID NO: 431 is the determined cDNA sequence for clone 27632.

SEQ ID NO: 432 is the determined cDNA sequence for clone 27633.

SEQ ID NO: 433 is the determined cDNA sequence for clone 27635.

SEQ ID NO: 434 is the determined cDNA sequence for clone 27636.

SEQ ID NO: 435 is the determined cDNA sequence for clone 27637.

SEQ ID NO: 436 is the determined cDNA sequence for clone 27638.

SEQ ID NO: 437 is the determined cDNA sequence for clone 27639.

SEQ ID NO: 438 is the determined cDNA sequence for clone 27640.

SEQ ID NO: 439 is the determined cDNA sequence for clone 27641.

SEQ ID NO: 440 is the determined CDNA sequence for clone 27642.

SEQ ID NO: 441 is the determined cDNA sequence for clone 27644.

SEQ ID NO: 442 is the determined cDNA sequence for clone 27646.

SEQ ID NO: 443 is the determined cDNA sequence for clone 27647.

SEQ ID NO: 444 is the determined cDNA sequence for clone 27649.

SEQ ID NO: 445 is the determined cDNA sequence for clone 27650.

SEQ ID NO: 446 is the determined cDNA sequence for clone 27651.

SEQ ID NO: 447 is the determined cDNA sequence for clone 27652.

SEQ ID NO: 448 is the determined cDNA sequence for clone 27654.

SEQ ID NO: 449 is the determined cDNA sequence for clone 27655.

SEQ ID NO: 450 is the determined cDNA sequence for clone 27657.

SEQ ID NO: 451 is the determined cDNA sequence for clone 27659.

SEQ ID NO: 452 is the determined cDNA sequence for clone 27665.

SEQ ID NO: 453 is the determined cDNA sequence for clone 27666.

SEQ ID NO: 454 is the determined cDNA sequence for clone 27668.

SEQ ID NO: 455 is the determined cDNA sequence for clone 27670.

SEQ ID NO: 456 is the determined cDNA sequence for clone 27671.

SEQ ID NO: 457 is the determined CDNA sequence for clone 27672.

SEQ ID NO: 458 is the determined cDNA sequence for clone 27674.

SEQ ID NO: 459 is the determined cDNA sequence for clone 27677.

SEQ ID NO: 460 is the determined cDNA sequence for clone 27681.

SEQ ID NO: 461 is the determined cDNA sequence for clone 27682.

SEQ ID NO: 462 is the determined cDNA sequence for clone 27683.

SEQ ID NO: 463 is the determined cDNA sequence for clone 27686.

SEQ ID NO: 464 is the determined cDNA sequence for clone 27688.

SEQ ID NO: 465 is the determined cDNA sequence for clone 27689.

SEQ ID NO: 466 is the determined cDNA sequence for clone 27690.

SEQ ID NO: 467 is the determined cDNA sequence for clone 27693.

SEQ ID NO: 468 is the determined cDNA sequence for clone 27699.

SEQ ID NO: 469 is the determined cDNA sequence for clone 27700.

SEQ ID NO: 470 is the determined cDNA sequence for clone 27702.

SEQ ID NO: 471 is the determined cDNA sequence for clone 27705.

SEQ ID NO: 472 is the determined cDNA sequence for clone 27706.

SEQ ID NO: 473 is the determined cDNA sequence for clone 27707.

SEQ ID NO: 474 is the determined cDNA sequence for clone 27708.

SEQ ID NO: 475 is the determined cDNA sequence for clone 27709.

SEQ ID NO: 476 is the determined cDNA sequence for clone 27710.

SEQ ID NO: 477 is the determined cDNA sequence for clone 27711.

SEQ ID NO: 478 is the determined cDNA sequence for clone 27712.

SEQ ID NO: 479 is the determined cDNA sequence for clone 27713.

SEQ ID NO: 480 is the determined cDNA sequence for clone 27714.

SEQ ID NO: 481 is the determined cDNA sequence for clone 27715.

SEQ ID NO: 482 is the determined cDNA sequence for clone 27716.

SEQ ID NO: 483 is the determined cDNA sequence for clone 27717.

SEQ ID NO: 484 is the determined cDNA sequence for clone 27718.

SEQ ID NO: 485 is the determined cDNA sequence for clone 27719.

SEQ ID NO: 486 is the determined cDNA sequence for clone 27720.

SEQ ID NO: 487 is the determined cDNA sequence for clone 27722.

SEQ ID NO: 488 is the determined cDNA sequence for clone 27723.

SEQ ID NO: 489 is the determined cDNA sequence for clone 27724.

SEQ ID NO: 490 is the determined cDNA sequence for clone 27726.

SEQ ID NO: 491 is the determined cDNA sequence for clone 25015.

SEQ ID NO: 492 is the determined cDNA sequence for clone 25016.

SEQ ID NO: 493 is the determined cDNA sequence for clone 25017.

SEQ ID NO: 494 is the determined cDNA sequence for clone 25018

SEQ ID NO: 495 is the determined cDNA sequence for clone 25030.

SEQ ID NO: 496 is the determined cDNA sequence for clone 25033.

SEQ ID NO: 497 is the determined cDNA sequence for clone 25034.

SEQ ID NO: 498 is the determined cDNA sequence for clone 25035.

SEQ ID NO: 499 is the determined cDNA sequence for clone 25036.

SEQ ID NO: 500 is the determined cDNA sequence for clone 25037.

SEQ ID NO: 501 is the determined cDNA sequence for clone 25038.

SEQ ID NO: 502 is the determined cDNA sequence for clone 25039.

SEQ ID NO: 503 is the determined cDNA sequence for clone 25040.

SEQ ID NO: 504 is the determined cDNA sequence for clone 25042.

SEQ ID NO: 505 is the determined cDNA sequence for clone 25043.

SEQ ID NO: 506 is the determined cDNA sequence for clone 25044.

SEQ ID NO: 507 is the determined cDNA sequence for clone 25045.

SEQ ID NO: 508 is the determined cDNA sequence for clone 25047.

SEQ ID NO: 509 is the determined cDNA sequence for clone 25048.

SEQ ID NO: 510 is the determined cDNA sequence for clone 25049.

SEQ ID NO: 511 is the determined cDNA sequence for clone 25185.

SEQ ID NO: 512 is the determined cDNA sequence for clone 25186.

SEQ ID NO: 513 is the determined cDNA sequence for clone 25187.

SEQ ID NO: 514 is the determined cDNA sequence for clone 25188.

SEQ ID NO: 515 is the determined cDNA sequence for clone 25189.

SEQ ID NO: 516 is the determined cDNA sequence for clone 25190.

SEQ ID NO: 517 is the determined cDNA sequence for clone 25193.

SEQ ID NO: 518 is the determined cDNA sequence for clone 25194.

SEQ ID NO: 519 is the determined cDNA sequence for clone 25196.

SEQ ID NO: 520 is the determined cDNA sequence for clone 25198.

SEQ ID NO: 521 is the determined cDNA sequence for clone 25199.

SEQ ID NO: 522 is the determined cDNA sequence for clone 25200.

SEQ ID NO: 523 is the determined cDNA sequence for clone 25202.

SEQ ID NO: 524 is the determined cDNA sequence for clone 25364.

SEQ ID NO: 525 is the determined cDNA sequence for clone 25366.

SEQ ID NO: 526 is the determined cDNA sequence for clone 25367.

SEQ ID NO: 527 is the determined cDNA sequence for clone 25368.

SEQ ID NO: 528 is the determined cDNA sequence for clone 25369.

SEQ ID NO: 529 is the determined cDNA sequence for clone 25370.

SEQ ID NO: 530 is the determined cDNA sequence for clone 25371.

SEQ ID NO: 531 is the determined cDNA sequence for clone 25372.

SEQ ID NO: 532 is the determined cDNA sequence for clone 25373.

SEQ ID NO: 533 is the determined cDNA sequence for clone 25374.

SEQ ID NO: 534 is the determined cDNA sequence for clone 25376.

SEQ ID NO: 535 is the determined cDNA sequence for clone 25377.

SEQ ID NO: 536 is the determined cDNA sequence for clone 25378.

SEQ ID NO: 537 is the determined cDNA sequence for clone 25379.

SEQ ID NO: 538 is the determined cDNA sequence for clone 25380.

SEQ ID NO: 539 is the determined cDNA sequence for clone 25381.

SEQ ID NO: 540 is the determined cDNA sequence for clone 25382.

SEQ ID NO: 541 is the determined cDNA sequence for clone 25383.

SEQ ID NO: 542 is the determined cDNA sequence for clone 25385.

SEQ ID NO: 543 is the determined cDNA sequence for clone 25386.

SEQ ID NO: 544 is the determined cDNA sequence for clone 25387.

SEQ ID NO: 545 is the determined cDNA sequence for clone 26013.

SEQ ID NO: 546 is the determined cDNA sequence for clone 26014.

SEQ ID NO: 547 is the determined cDNA sequence for clone 26016.

SEQ ID NO: 548 is the determined cDNA sequence for clone 26017.

SEQ ID NO: 549 is the determined cDNA sequence for clone 26018.

SEQ ID NO: 550 is the determined cDNA sequence for clone 26019.

SEQ ID NO: 551 is the determined cDNA sequence for clone 26020.

SEQ ID NO: 552 is the determined cDNA sequence for clone 26021.

SEQ ID NO: 553 is the determined CDNA sequence for clone 26022.

SEQ ID NO: 554 is the determined cDNA sequence for clone 26027.

SEQ ID NO: 555 is the determined cDNA sequence for clone 26197.

SEQ ID NO: 556 is the determined cDNA sequence for clone 26199.

SEQ ID NO: 557 is the determined cDNA sequence for clone 26201.

SEQ ID NO: 558 is the determined cDNA sequence for clone 26202.

SEQ ID NO: 559 is the determined cDNA sequence for clone 26203.

SEQ ID NO: 560 is the determined cDNA sequence for clone 26204.

SEQ ID NO: 561 is the determined cDNA sequence for clone 26205.

SEQ ID NO: 562 is the determined cDNA sequence for clone 26206.

SEQ ID NO: 563 is the determined cDNA sequence for clone 26208.

SEQ ID NO: 564 is the determined cDNA sequence for clone 26211.

SEQ ID NO: 565 is the determined cDNA sequence for clone 26212.

SEQ ID NO: 566 is the determined cDNA sequence for clone 26213.

SEQ ID NO: 567 is the determined cDNA sequence for clone 26214.

SEQ ID NO: 568 is the determined cDNA sequence for clone 26215.

SEQ ID NO: 569 is the determined .DNA sequence for clone 26216.

SEQ ID NO: 570 is the determined cDNA sequence for clone 26217.

SEQ ID NO: 571 is the determined cDNA sequence for clone 26218.

SEQ ID NO: 572 is the determined cDNA sequence for clone 26219.

SEQ ID NO: 573 is the determined cDNA sequence for clone 26220.

SEQ ID NO: 574 is the determined cDNA sequence for clone 26221.

SEQ ID NO: 575 is the determined cDNA sequence for clone 26224.

SEQ ID NO: 576 is the determined cDNA sequence for clone 26225.

SEQ ID NO: 577 is the determined cDNA sequence for clone 26226.

SEQ ID NO: 578 is the determined cDNA sequence for clone 26227.

SEQ ID NO: 579 is the determined cDNA sequence for clone 26228.

SEQ ID NO: 580 is the determined cDNA sequence for clone 26230.

SEQ ID NO: 581 is the determined cDNA sequence for clone 26231.

SEQ ID NO: 582 is the determined CDNA sequence for clone 26234.

SEQ ID NO: 583 is the determined cDNA sequence for clone 26236.

SEQ ID NO: 584 is the determined cDNA sequence for clone 26237.

SEQ ID NO: 585 is the determined cDNA sequence for clone 26239.

SEQ ID NO: 586 is the determined cDNA sequence for clone 26240.

SEQ ID NO: 587 is the determined cDNA sequence for clone 26241.

SEQ ID NO: 588 is the determined cDNA sequence for clone 26242.

SEQ ID NO: 589 is the determined cDNA sequence for clone 26246.

SEQ ID NO: 590 is the determined cDNA sequence for clone 26247.

SEQ ID NO: 591 is the determined cDNA sequence for clone 26248.

SEQ ID NO: 592 is the determined cDNA sequence for clone 26249.

SEQ ID NO: 593 is the determined cDNA sequence for clone 26250.

SEQ ID NO: 594 is the determined cDNA sequence for clone 26251.

SEQ ID NO, 595 is the determined cDNA sequence for clone 26252.

SEQ ID NO: 596 is the determined cDNA sequence for clone 26253.

SEQ ID NO: 597 is the determined cDNA sequence for clone 26254.

SEQ ID NO: 598 is the determined cDNA sequence for clone 26255.

SEQ ID NO: 599 is the determined cDNA sequence for clone 26256.

SEQ ID NO: 600 is the determined cDNA sequence for clone 26257.

SEQ ID NO: 601 is the determined cDNA sequence for clone 26259.

SEQ ID NO: 602 is the determined cDNA sequence for clone 26260.

SEQ ID NO: 603 is the determined cDNA sequence for clone 26261.

SEQ ID NO: 604 is the determined cDNA sequence for clone 26262.

SEQ ID NO: 605 is the determined cDNA sequence for clone 26263.

SEQ ID NO: 606 is the determined cDNA sequence for clone 26264.

SEQ ID NO: 607 is the determined cDNA sequence for clone 26265.

SEQ ID NO: 608 is the determined cDNA sequence for clone 26266.

SEQ ID NO: 609 is the determined cDNA sequence for clone 26268.

SEQ ID NO: 610 is the determined cDNA sequence for clone 26269.

SEQ ID NO: 611 is the determined CDNA sequence for clone 26271.

SEQ ID NO: 612 is the determined cDNA sequence for clone 26273.

SEQ ID NO: 613 is the determined cDNA sequence for clone 26810.

SEQ ID NO: 614 is the determined cDNA sequence for clone 26811.

SEQ ID NO: 615 is the determined cDNA sequence for clone 26812.1.

SEQ ID NO: 616 is the determined cDNA sequence for clone 26812.2.

SEQ ID NO: 617 is the determined cDNA sequence for clone 26813.

SEQ ID NO: 618 is the determined cDNA sequence for clone 26814.

SEQ ID NO: 619 is the determined cDNA sequence for clone 26815.

SEQ ID NO: 620 is the determined cDNA sequence for clone 26816.

SEQ ID NO: 621 is the determined cDNA sequence for clone 26818.

SEQ ID NO: 622 is the determined cDNA sequence for clone 26819.

SEQ ID NO: 623 is the determined cDNA sequence for clone 26820.

SEQ ID NO: 624 is the determined cDNA sequence for clone 26821.

SEQ ID NO: 625 is the determined cDNA sequence for clone 26822.

SEQ ID NO: 626 is the determined cDNA sequence for clone 26824.

SEQ ID NO: 627 is the determined CDNA sequence for clone 26825.

SEQ ID NO: 628 is the determined cDNA sequence for clone 26826.

SEQ ID NO: 629 is the determined cDNA sequence for clone 26827.

SEQ ID NO: 630 is the determined CDNA sequence for clone 26829.

SEQ ID NO: 631 is the determined ;DNA sequence for clone 26830.

SEQ ID NO: 632 is the determined cDNA sequence for clone 26831.

SEQ ID NO: 633 is the determined cDNA sequence for clone 26832.

SEQ ID NO: 634 is the determined cDNA sequence for clone 26835.

SEQ ID NO: 635 is the determined ,DNA sequence for clone 26836.

SEQ ID NO: 636 is the determined cDNA sequence for clone 26837.

SEQ ID NO: 637 is the determined cDNA sequence for clone 26839.

SEQ ID NO: 638 is the determined cDNA sequence for clone 26841.

SEQ ID NO: 639 is the determined cDNA sequence for clone 26843.

SEQ ID NO: 640 is the determined cDNA sequence for clone 26844.

SEQ ID NO: 641 is the determined cDNA sequence for clone 26845.

SEQ ID NO: 642 is the determined cDNA sequence for clone 26846.

SEQ ID NO: 643 is the determined cDNA sequence for clone 26847.

SEQ ID NO: 644 is the determined cDNA sequence for clone 26848.

SEQ ID NO: 645 is the determined cDNA sequence for clone 26849.

SEQ ID NO: 646 is the determined cDNA sequence for clone 26850.

SEQ ID NO: 647 is the determined cDNA sequence for clone 26851.

SEQ ID NO: 648 is the determined cDNA sequence for clone 26852.

SEQ ID NO: 649 is the determined cDNA sequence for clone 26853.

SEQ ID NO: 650 is the determined cDNA sequence for clone 26854.

SEQ ID NO: 651 is the determined cDNA sequence for clone 26856.

SEQ ID NO: 652 is the determined cDNA sequence for clone 26857.

SEQ ID NO: 653 is the determined cDNA sequence for clone 26858.

SEQ ID NO: 654 is the determined cDNA sequence for clone 26859.

SEQ ID NO: 655 is the determined cDNA sequence for clone 26860.

SEQ ID NO: 656 is the determined cDNA sequence for clone 26862.

SEQ ID NO: 657 is the determined cDNA sequence for clone 26863.

SEQ ID NO: 658 is the determined cDNA sequence for clone 26864.

SEQ ID NO: 659 is the determined cDNA sequence for clone 26865.

SEQ ID NO: 660 is the determined cDNA sequence for clone 26867.

SEQ ID NO: 661 is the determined cDNA sequence for clone 26868.

SEQ ID NO: 662 is the determined cDNA sequence for clone 26871.

SEQ ID NO: 663 is the determined cDNA sequence for clone 26873.

SEQ ID NO: 664 is the determined cDNA sequence for clone 26875.

SEQ ID NO: 665 is the determined cDNA sequence for clone 26876.

SEQ ID NO: 666 is the determined cDNA sequence for clone 26877.

SEQ ID NO: 667 is the determined cDNA sequence for clone 26878.

SEQ ID NO: 668 is the determined cDNA sequence for clone 26880.

SEQ ID NO: 669 is the determined cDNA sequence for clone 26882.

SEQ ID NO: 670 is the determined cDNA sequence for clone 26883.

SEQ ID NO: 671 is the determined cDNA sequence for clone 26884.

SEQ ID NO: 672 is the determined cDNA sequence for clone 26885.

SEQ ID NO: 673 is the determined cDNA sequence for clone 26886.

SEQ ID NO: 674 is the determined cDNA sequence for clone 26887.

SEQ ID NO: 675 is the determined cDNA sequence for clone 26888.

SEQ ID NO: 676 is the determined cDNA sequence for clone 26889.

SEQ ID NO: 677 is the determined cDNA sequence for clone 26890.

SEQ ID NO: 678 is the determined cDNA sequence for clone 26892.

SEQ ID NO: 679 is the determined cDNA sequence for clone 26894.

SEQ ID NO: 680 is the determined cDNA sequence for clone 26895.

SEQ ID NO: 681 is the determined cDNA sequence for clone 26897.

SEQ ID NO: 682 is the determined cDNA sequence for clone 26898.

SEQ ID NO: 683 is the determined cDNA sequence for clone 26899.

SEQ ID NO: 684 is the determined cDNA sequence for clone 26900.

SEQ ID NO: 685 is the determined cDNA sequence for clone 26901.

SEQ ID NO: 686 is the determined cDNA sequence for clone 26903.

SEQ ID NO: 687 is the determined cDNA sequence for clone 26905.

SEQ ID NO: 688 is the determined cDNA sequence for clone 26906.

SEQ ID NO: 689 is the determined cDNA sequence for clone 26708.

SEQ ID NO: 690 is the determined cDNA sequence for clone 26709.

SEQ ID NO: 691 is the determined cDNA sequence for clone 26710.

SEQ ID NO: 692 is the determined cDNA sequence for clone 26711.

SEQ ID NO: 693 is the determined cDNA sequence for clone 26712.

SEQ ID NO: 694 is the determined cDNA sequence for clone 26713.

SEQ ID NO: 695 is the determined cDNA sequence for clone 26714.

SEQ ID NO: 696 is the determined cDNA sequence for clone 26715.

SEQ ID NO: 697 is the determined cDNA sequence for clone 26716.

SEQ ID NO: 698 is the determined cDNA sequence for clone 26717.

SEQ ID NO: 699 is the determined cDNA sequence for clone 26718.

SEQ ID NO: 700 is the determined cDNA sequence for clone 26719.

SEQ ID NO: 701 is the determined cDNA sequence for clone 26720.

SEQ ID NO: 702 is the determined cDNA sequence for clone 26721.

SEQ ID NO: 703 is the determined cDNA sequence for clone 26722.

SEQ ID NO: 704 is the determined cDNA sequence for clone 26723.

SEQ ID NO: 705 is the determined cDNA sequence for clone 26724.

SEQ ID NO: 706 is the determined cDNA sequence for clone 26725.

SEQ ID NO: 707 is the determined cDNA sequence for clone 26726.

SEQ ID NO: 708 is the determined cDNA sequence for clone 26727.

SEQ ID NO: 709 is the determined cDNA sequence for clone 26728.

SEQ ID NO: 710 is the determined cDNA sequence for clone 26729.

SEQ ID NO: 711 is the determined cDNA sequence for clone 26730.

SEQ ID NO: 712 is the determined cDNA sequence for clone 26731.

SEQ ID NO: 713 is the determined cDNA sequence for clone 26732.

SEQ ID NO: 714 is the determined cDNA sequence for clone 26733.1.

SEQ ID NO: 715 is the determined cDNA sequence for clone 26733.2.

SEQ ID NO: 716 is the determined cDNA sequence for clone 26734.

SEQ ID NO: 717 is the determined cDNA sequence for clone 26735.

SEQ ID NO: 718 is the determined cDNA sequence for clone 26736.

SEQ ID NO: 719 is the determined cDNA sequence for clone 26737.

SEQ ID NO: 720 is the determined cDNA sequence for clone 26738.

SEQ ID NO: 721 is the determined cDNA sequence for clone 26739.

SEQ ID NO: 722 is the determined cDNA sequence for clone 26741.

SEQ ID NO: 723 is the determined cDNA sequence for clone 26742.

SEQ ID NO: 724 is the determined cDNA sequence for clone 26743.

SEQ ID NO: 725 is the determined cDNA sequence for clone 26744.

SEQ ID NO: 726 is the determined cDNA sequence for clone 26745.

SEQ ID NO: 727 is the determined cDNA sequence for clone 26746.

SEQ ID NO: 728 is the determined cDNA sequence for clone 26747.

SEQ ID NO: 729 is the determined cDNA sequence for clone 26748.

SEQ ID NO: 730 is the determined cDNA sequence for clone 26749.

SEQ ID NO: 731 is the determined cDNA sequence for clone 26750.

SEQ ID NO: 732 is the determined cDNA sequence for clone 26751.

SEQ ID NO: 733 is the determined cDNA sequence for clone 26752.

SEQ ID NO: 734 is the determined cDNA sequence for clone 26753.

SEQ ID NO: 735 is the determined cDNA sequence for clone 26754.

SEQ ID NO: 736 is the determined cDNA sequence for clone 26755.

SEQ ID NO: 737 is the determined cDNA sequence for clone 26756.

SEQ ID NO: 738 is the determined cDNA sequence for clone 26757.

SEQ ID NO: 739 is the determined cDNA sequence for clone 26758.

SEQ ID NO: 740 is the determined cDNA sequence for clone 26759.

SEQ ID NO: 741 is the determined cDNA sequence for clone 26760.

SEQ ID NO: 742 is the determined cDNA sequence for clone 26761.

SEQ ID NO: 743 is the determined cDNA sequence for clone 26762.

SEQ ID NO: 744 is the determined cDNA sequence for clone 26763.

SEQ ID NO: 745 is the determined cDNA sequence for clone 26764.

SEQ ID NO: 746 is the determined cDNA sequence for clone 26765.

SEQ ID NO: 747 is the determined cDNA sequence for clone 26766.

SEQ ID NO: 748 is the determined cDNA sequence for clone 26767.

SEQ ID NO: 749 is the determined cDNA sequence for clone 26768.

SEQ ID NO: 750 is the determined cDNA sequence for clone 26769.

SEQ ID NO: 751 is the determined cDNA sequence for clone 26770.

SEQ ID NO: 752 is the determined cDNA sequence for clone 26771.

SEQ ID NO: 753 is the determined cDNA sequence for clone 26772.

SEQ ID NO: 754 is the determined cDNA sequence for clone 26773.

SEQ ID NO: 755 is the determined cDNA sequence for clone 26774.

SEQ ID NO: 756 is the determined cDNA sequence for clone 26775.

SEQ ID NO: 757 is the determined cDNA sequence for clone 26776.

SEQ ID NO: 758 is the determined cDNA sequence for clone 26777.

SEQ ID NO: 759 is the determined cDNA sequence for clone 26778.

SEQ ID NO: 760 is the determined cDNA sequence for clone 26779.

SEQ ID NO: 761 is the determined cDNA sequence for clone 26781.

SEQ ID NO: 762 is the determined cDNA sequence for clone 26782.

SEQ ID NO: 763 is the determined cDNA sequence for clone 26783.

SEQ ID NO: 764 is the determined cDNA sequence for clone 26784.

SEQ ID NO: 765 is the determined cDNA sequence for clone 26785.

SEQ ID NO: 766 is the determined cDNA sequence for clone 26786.

SEQ ID NO: 767 is the determined cDNA sequence for clone 26787.

SEQ ID NO: 768 is the determined cDNA sequence for clone 26788.

SEQ ID NO: 769 is the determined cDNA sequence for clone 26790.

SEQ ID NO: 770 is the determined cDNA sequence for clone 26791.

SEQ ID NO: 771 is the determined cDNA sequence for clone 26792.

SEQ ID NO: 772 is the determined cDNA sequence for clone 26793.

SEQ ID NO: 773 is the determined cDNA sequence for clone 26794.

SEQ ID NO: 774 is the determined cDNA sequence for clone 26795.

SEQ ID NO: 775 is the determined cDNA sequence for clone 26796.

SEQ ID NO: 776 is the determined cDNA sequence for clone 26797.

SEQ ID NO: 777 is the determined cDNA sequence for clone 26798.

SEQ ID NO: 778 is the determined cDNA sequence for clone 26800.

SEQ ID NO: 779 is the determined cDNA sequence for clone 26801.

SEQ ID NO: 780 is the determined cDNA sequence for clone 26802.

SEQ ID NO: 781 is the determined cDNA sequence for clone 26803.

SEQ ID NO: 782 is the determined cDNA sequence for clone 26804.

SEQ ID NO: 783 is the amino acid sequence for L773P.

SEQ ID NO: 784 is the determined DNA sequence of the L773P expressionconstruct.

SEQ ID NO: 785 is the determined DNA sequence of the L773PA expressionconstruct.

SEQ ID NO: 786 is a predicted amino acid sequence for L552S.

SEQ ID NO: 787 is a predicted amino acid sequence for L840P.

SEQ ID NO: 788 is the full-length cDNA sequence for L548S.

SEQ ID NO: 789 is the amino acid sequence encoded by

SEQ ID NO: 788.

SEQ ID NO: 790 is an extended cDNA sequence for L552S.

SEQ ID NO: 791 is the predicted amino acid sequence encoded by the cDNAsequence of

SEQ ID NO: 790.

SEQ ID NO: 792 is the determined cDNA sequence for an isoform of L552S.

SEQ ID NO: 793 is the predicted amino acid sequence encoded by SEQ IDNO: 792.

SEQ ID NO: 794 is an extended cDNA sequence for L840P.

SEQ ID NO: 795 is the predicted amino acid sequence encoded by SEQ DINO: 794.

SEQ ID NO: 796 is an extended cDNA sequence for L801P.

SEQ ID NO: 797 is a first predicted amino acid sequence encoded by SEQID NO: 796.

SEQ ID NO: 798 is a second predicted amino acid sequence encoded by SEQID NO: 796.

SEQ ID NO: 799 is a third predicted amino acid sequence encoded by SEQID NO: 796.

SEQ ID NO: 800 is the determined full-length sequence for L844P.

SEQ ID NO: 801 is the 5′ consensus cDNA sequence for L551S.

SEQ ID NO: 802 is the 3′ consensus cDNA sequence for L551 S.

SEQ ID NO: 803 is the cDNA sequence for STY8.

SEQ ID NO: 804 is an extended cDNA sequence for L551S.

SEQ ID NO: 805 is the amino acid sequence for STY8.

SEQ ID NO: 806 is the extended amino acid sequence for L551S.

SEQ ID NO: 807 is the determined full-length CDNA sequence for L773P.

SEQ ID NO: 808 is the full-length CDNA sequence of L552S.

SEQ ID NO: 809 is the full-length amino acid sequence of L552S.

SEQ ID NO: 810 is the determined cDNA sequence of clone 50989.

SEQ ID NO: 811 is the determined cDNA sequence of clone 50990.

SEQ ID NO: 812 is the determined cDNA sequence of clone 50992.

SEQ ID NO: 813-824 are the determined cDNA sequences for clones isolatedfrom lung tumor tissue.

SEQ ID NO: 825 is the determined cDNA sequence for the full-length L551Sclone 54305.

SEQ ID NO: 826 is the determined cDNA sequence for the full-length L551Sclone 54298.

SEQ ID NO: 827 is the full-length amino acid sequence for L551S.

Tables 1-6 contain the sequence identifiers for SEQ ID NO:878-1664.

TABLE 1A SEQ ID CLONE NO IDENTIFIER 828 R0126:A02 829 R0126:A03 830R0126:A05 831 R0126:A06 832 R0126:A08 833 R0126:A09 834 R0126:A10 835R0126:A11 836 R0126:A12 837 R0126:B01 838 R0126:B03 839 R0126:B04 840R0126:B05 841 R0126:B06 842 R0126:B07 843 R0126:B08 844 R0126:B09 845R0126:B11 846 R0126:B12 847 R0126:C01 848 R0126:C02 849 R0126:C03 850R0126:C05 851 R0126:C06 852 R0126:C07 853 R0126:C08 854 R0126:C09 855R0126:C10 856 R0126:C11 857 R0126:C12 858 R0126:D01 859 R0126:D02 860R0126:D03 861 R0126:D04 862 R0126:D05 863 R0126:D06 864 R0126:D07 865R0126:D08 866 R0126:D09 867 R0126:D10 868 R0126:D11 869 R0126:D12 870R0126:E01 871 R0126:E02 872 R0126:E03 873 R0126:E04 874 R0126:E05 875R0126:E06 876 R0126:E07 877 R0126:E08 878 R0126:E09 879 R0126:E10 880R0126:E11 881 R0126:E12 882 R0126:F01 883 R0126:F02 884 R0126:F03 885R0126:F04 886 R0126:F05 887 R0126:F06 888 R0126:F07 889 R0126:F08 890R0126:F10 891 R0126:F11 892 R0126:F12 893 R0126:G01 894 R0126:G02 895R0126:G03 896 R0126:G04 897 R0126:G05 898 R0126:G06 899 R0126:G07 900R0126:G09 901 R0126:G10 902 R0126:G11 903 R0126:G12 904 R0126:H01 905R0126:H02 906 R0126:H03 907 R0126:H04 908 R0126:H05 909 R0126:H06

TABLE 1B SEQ ID CLONE NO IDENTIFIER 910 R0126:H07 911 R0126:H09 912R0126:H10 913 R0126:H11 914 R0127:A02 915 R0127:A05 916 R0127:A06 917R0127:A07 918 R0127:A08 919 R0127:A09 920 R0127:A10 921 R0127:A11 922R0127:A12 923 R0127:B01 924 R0127:B03 925 R0127:B04 926 R0127:B05 927R0127:B06 928 R0127:B07 929 R0127:B08 930 R0127:B09 931 R0127:B10 932R0127:B11 933 R0127:B12 934 R0127:C01 935 R0127:C03 936 R0127:C04 937R0127:C05 938 R0127:C07 939 R0127:C08 940 R0127:C09 941 R0127:C10 942R0127:C11 943 R0127:D01 944 R0127:D02 945 R0127:D03 946 R0127:D04 947R0127:D05 948 R0127:D06 949 R0127:D07 950 R0127:D01 951 R0127:D10 952R0127:D11 953 R0127:D12 954 R0127:E02 955 R0127:E03 956 R0127:E04 957R0127:E05 958 R0127:E06 959 R0127:E07 960 R0127:E08 961 R0127:E09 962R0127:E10 963 R0127:E11 964 R0127:F01 965 R0127:F02 966 R0127:F03 967R0127:F04 968 R0127:F05 969 R0127:F06 970 R0127:F07 971 R0127:F08 972R0127:F10 973 R0127:F11 974 R0127:F12 975 R0127:G01 976 R0127:G02 977R0127:G03 978 R0127:G04 979 R0127:G05 980 R0127:G06 981 R0127:G07 982R0127:G08 983 R0127:G09 984 R0127:G10 985 R0127:G11 986 R0127:G12 987R0127:H01 988 R0127:H02 989 R0127:H03 990 R0127:H04 991 R0127:H05

TABLE 1C SEQ ID CLONE NO IDENTIFIER  992 R0127:H06  993 R0127:H07  994R0127:H08  995 R1027:H09  996 R1027:H10  997 R1027:H11  998 R1028:A02 999 R1028:A05 1000 R1028:A06 1001 R1028:A07 1002 R1028:A08 1003R1028:A09 1004 R1028:A10 1005 R1028:B01 1006 R1028:B02 1007 R1028:B031008 R1028:B04 1009 R1028:B05 1010 R1028:B08 1011 R1028:B09 1012R1028:B10 1013 R1028:B11 1014 R1028:B12 1015 R1028:C01 1016 R1028:C031017 R1028:C04 1018 R1028:C05 1019 R1028:C06 1020 R1028:C07 1021R1028:C08 1022 R1028:C10 1023 R1028:C11 1024 R1028:C12 1025 R1028:D011026 R1028:D02 1027 R1028:D04 1028 R1028:D05 1029 R1028:D06 1030R1028:D07 1031 R1028:D08 1032 R1028:D09 1033 R0128:D10 1034 R0128:D111035 R0128:D12 1036 R0128:E01 1037 R0128:E02 1038 R0128:E03 1039R0128:E04 1040 R0128:E05 1041 R0128:E06 1042 R0128:E07 1043 R0128:E081044 R0128:E09 1045 R0128:E10 1046 R0128:E12 1047 R0128:F01 1048R0128:F02 1049 R0128:F03 1050 R0128:F04 1051 R0128:F06 1052 R0128:F071053 R0128:F08 1054 R0128:F09 1055 R0128:F10 1056 R0128:F12 1057R0128:G01 1058 R0128:G02 1059 R0128:G03 1060 R0128:G04 1061 R0128:G051062 R0128:G06 1063 R0128:G07 1064 R0128:G09 1065 R0128:G10 1066R0128:G11 1067 R0128:G12 1068 R0128:H01 1069 R0128:H02 1070 R0128:H031071 R0128:H04 1072 R0128:H05 1073 R0128:H06 1074 R0128:H07 1075R0128:H08

TABLE 1D SEQ ID CLONE NO IDENTIFIER 1076 R0128:H09 1077 R0128:H10 1078R0128:H11 1079 R0130:A02 1080 R0130:A05 1081 R0130:A06 1082 R0130:A081083 R0130:A09 1084 R0130:A10 1085 R0130:A11 1086 R0130:A12 1087R0130:B01 1088 R0130:B02 1089 R0130:B03 1090 R0130:B04 1091 R0130:B051092 R0130:B06 1093 R0130:B08 1094 R0130:B09 1095 R0130:B10 1096R0130:B11 1097 R0130:B12 1098 R0130:C02 1099 R0130:C03 1100 R0130:C041101 R0130:C05 1102 R0130:C06 1103 R0130:C07 1104 R0130:C08 1105R0130:C09 1106 R0130:C10 1107 R0130:C11 1108 R0130:C12 1109 R0130:D021110 R0130:D03 1111 R0130:D04 1112 R0130:D05 1113 R0130:D06 1114R0130:D07 1115 R0130:D09 1116 R0130:D10 1117 R0130:D11 1118 R0130:D121119 R0130:E01 1120 R0130:E02 1121 R0130:E03 1122 R0130:E04 1123R0130:E05 1124 R0130:E06 1125 R0130:E07 1126 R0130:E08 1127 R0130:E091128 R0130:E10 1129 R0130:E11 1130 R0130:E12 1131 R0130:F02 1132R0130:F03 1133 R0130:F05 1134 R0130:F06 1135 R0130:F07 1136 R0130:F081137 R0130:F09 1138 R0130:F10 1139 R0130:F11 1140 R0130:F12 1141R0130:G01 1142 R0130:G02 1143 R0130:G03 1144 R0130:G04 1145 R0130:G051146 R0130:G06 1147 R0130:G07 1148 R0130:G08 1149 R0130:G09 1150R0130:G10 1151 R0130:G11 1152 R0130:G12 1153 R0130:H01 1154 R0130:H021155 R0130:H04 1156 R0130:H05 1157 R0130:H06 1158 R0130:H07 1159R0130:H08

TABLE 1E SEQ ID CLONE NO IDENTIFIER 1160 R0130:H09 1161 R0130:H10 1162R0130:H11 1163 R0131:A02 1164 R0131:A05 1165 R0131:A06 1166 R0131:A071167 R0131:A08 1168 R0131:A09 1169 R0131:A11 1170 R0131:A12 1171R0131:B02 1172 R0131:B03 1173 R0131:B04 1174 R0131:B05 1175 R0131:B071176 R0131:B08 1177 R0131:B09 1178 R0131:B10 1179 R0131:B11 1180R0131:C01 1181 R0131:C02 1182 R0131:C03 1183 R0131:C04 1184 R0131:C061185 R0131:C07 1186 R0131:C08 1187 R0131:C10 1188 R0131:C11 1189R0131:C12 1190 R0131:D02 1191 R0131:D03 1192 R0131:D04 1193 R0131:D051194 R0131:D06 1195 R0131:D07 1196 R0131:D09 1197 R0131:D10 1198R0131:D11 1199 R0131:D12 1200 R0131:E01 1201 R0131:E02 1202 R0131:E031203 R0131:E04 1204 R0131:E06 1205 R0131:E07 1206 R0131:E08 1207R0131:E10 1208 R0131:E11 1209 R0131:E12 1210 R0131:F02 1211 R0131:F041212 R0131:F05 1213 R0131:F06 1214 R0131:F07 1215 R0131:F08 1216R0131:F09 1217 R0131:F10 1218 R0131:F11 1219 R0131:F12 1220 R0131:G011221 R0131:G02 1222 R0131:G03 1223 R0131:G04 1224 R0131:G05 1225R0131:G06 1226 R0131:G07 1227 R0131:G08 1228 R0131:G09 1229 R0131:G101230 R0131:G11 1231 R0131:G12 1232 R0131:H01 1233 R0131:H02 1234R0131:H05 1235 R0131:H06 1236 R0131:H07 1237 R0131:H08 1238 R0131:H091239 R0131:H11

TABLE 2 Clone names for NSCLC-SQL1 and corresponding SEQ ID NOs SEQ IDCLONE NO IDENTIFIER 1240 Contig 54 1241 Contig 55 1242 Contig 57 1243Contig 58 1244 Contig 60 1245 Contig 62 1246 Contig 63 1247 Contig 641248 Contig 65 1249 Contig 66 1250 Contig 67 1251 Contig 68 1252 Contig69 1253 Contig 70 1254 Contig 71 1255 Contig 72 1256 Contig 73 1257Contig 74 1258 Contig 75 1259 Contig 77 1260 Contig 78 1261 Contig 791262 Contig 80 1263 Contig 81 1264 Contig 83 1265 Contig 84 1266 Contig86 1267 Contig 87 1268 Contig 88 1269 Contig 89 1270 Contig 90 1271Contig 91 1272 Contig 92 1273 Contig 94 1274 Contig 95 1275 Contig 961276 Contig 97 1277 Contig 98 1278 Contig 99 1279  Contig 100

TABLE 3 Clone names for NSCLC-SCLI and corresponding SEQ ID NOs SEQ IDCLONE NO IDENTIFIER 1280 Contig 38 1281 Contig 39 1282 Contig 40 1283Contig 41 1284 Contig 42 1285 Contig 43 1286 Contig 44 1287 Contig 451288 Contig 46 1289 Contig 47 1290 Contig 48 1291 Contig 49 1292 Contig51 1293 Contig 52 1294 Contig 53 1295 Contig 54 1296 Contig 55 1297Contig 56 1298 Contig 57 1299 Contig 58 1300 Contig 59 1301 Contig 601302 Contig 62 1303 Contig 63 1304 Contig 64 1305 Contig 65 1306 Contig66 1307 Contig 67 1308 Contig 68 1309 Contig 69 1310 Contig 70 1311Contig 72 1312 Contig 73 1313 Contig 75 1314 Contig 76 1315 Contig 771316 Contig 78 1317 Contig 79 1318 Contig 80 1319 Contig 81 1320 Contig82

TABLE 4A Clone names for NSCLC-SCL3-SCL4 and corresponding SEQ ID NOsSEQ ID CLONE NO IDENTIFIER 1321 Contig 94  1322 Contig 95  1323 Contig96  1324 Contig 97  1325 Contig 98  1326 Contig 99  1327 Contig 100 1328Contig 101 1329 Contig 102 1330 Contig 103 1331 Contig 104 1332 Contig105 1333 Contig 106 1334 Contig 107 1335 Contig 108 1336 Contig 109 1337Contig 110 1338 Contig 111 1339 Contig 112 1340 Contig 113 1341 Contig114 1342 Contig 115 1343 Contig 116 1344 Contig 117 1345 Contig 118 1346Contig 119 1347 Contig 120 1348 Contig 121 1349 Contig 122 1350 Contig123 1351 Contig 124 1352 Contig 125 1353 Contig 126 1354 Contig 127 1355Contig 128 1356 Contig 129 1357 Contig 130 1358 Contig 131 1359 Contig132 1360 Contig 133 1361 Contig 134 1362 Contig 135 1363 Contig 136 1364Contig 137 1365 Contig 138 1366 Contig 139 1367 Contig 140 1368 Contig141 1369 Contig 142 1370 Contig 143 1371 Contig 144 1372 Contig 145 1373Contig 146 1374 Contig 147 1375 Contig 148 1376 Contig 149 1377 Contig150 1378 Contig 151 1379 Contig 152 1380 Contig 153 1381 Contig 154 1382Contig 155 1383 Contig 156 1384 Contig 157 1385 Contig 158 1386 Contig159 1387 Contig 160 1388 Contig 161 1389 Contig 162 1390 Contig 163 1391Contig 164 1392 Contig 165 1393 Contig 166 1394 Contig 167 1395 Contig168 1396 Contig 169 1397 Contig 170 1398 Contig 171 1399 Contig 172 1400Contig 173 1401 Contig 174 1402 Contig 175 1403 Contig 176

TABLE 4B Clone names for NSCLC-SCL3-SCL4 and corresponding SEQ ID NOsSEQ ID CLONE NO IDENTIFIER 1404 Contig 177 1405 Contig 178 1406 Contig179 1407 Contig 180 1408 Contig 181 1409 Contig 182 1410 Contig 183 1411Contig 184 1412 Contig 185 1413 Contig 186 1414 Contig 187

TABLE 5 Clone names for SCLC-SQL1 and corresponding SEQ ID NOs SEQ IDCLONE NO IDENTIFIER 1415 Contig 17 1416 Contig 18 1417 Contig 20 1418Contig 23 1419 Contig 24 1420 Contig 25 1421 Contig 26 1422 Contig 271423 Contig 28 1424 Contig 29 1425 Contig 30 1426 Contig 31 1427 Contig20 1428 Contig 21 1429 Contig 22 1430 Contig 23 1431 Contig 24 1432Contig 25 1433 Contig 26 1434 Contig 27 1435 Contig 28 1436 Contig 291437 Contig 30 1438 Contig 31 1439 Contig 32 1440 Contig 33 1441 Contig34 1442 Contig 35 1443 Contig 36 1444 Contig 37 1445 Contig 38

TABLE 6A Clone names for SCLC-SCL3-SCL4 and corresponding SEQ ID NOs SEQID CLONE NO IDENTIFIER 1446 Contig 116 1447 Contig 117 1448 Contig 1181449 Contig 119 1450 Contig 120 1451 Contig 122 1452 Contig 123 1453Contig 124 1454 Contig 125 1455 Contig 126 1456 Contig 127 1457 Contig128 1458 Contig 129 1459 Contig 130 1460 Contig 131 1461 Contig 132 1462Contig 133 1463 Contig 135 1464 Contig 136 1465 Contig 137 1466 Contig138 1467 Contig 139 (L985P) 1468 Contig 140 1469 Contig 141 1470 Contig142 1471 Contig 143 1472 Contig 144 1473 Contig 145 1474 Contig 146 1475Contig 147 1476 Contig 148 1477 Contig 149 1478 Contig 150 1479 Contig151 1480 Contig 152 1481 Contig 153 1482 Contig 154 1483 Contig 155 1484Contig 156 1485 Contig 157 1486 Contig 158 1487 Contig 159 1488 Contig160 1489 Contig 161 1490 Contig 162 1491 Contig 163 1492 Contig 164 1493Contig 165 1494 Contig 166 1495 Contig 167 1496 Contig 168 1497 Contig169 1498 Contig 170 1499 Contig 171 1500 Contig 172 1501 Contig 173 1502Contig 174 1503 Contig 175 1504 Contig 176 1505 Contig 177 1506 Contig178 1507 Contig 179 1508 Contig 181 1509 Contig 182 1510 Contig 183 1511Contig 184 1512 Contig 185 1513 Contig 186 1514 Contig 187 1515 Contig189 1516 Contig 190 1517 Contig 191 1518 Contig 192 1519 Contig 193 1520Contig 194 1521 Contig 195 1522 Contig 196 1523 Contig 197 1524 Contig198 1525 Contig 199 1526 Contig 200 1527 Contig 201 1528 Contig 202

TABLE 6B Clone names for SCLC-SCL3-SCL4 and corresponding SEQ ID NOs SEQID CLONE NO IDENTIFIER 1529 Contig 203 1530 Contig 204 1531 Contig 2051532 Contig 206 1533 Contig 207 1534 Contig 208 1535 Contig 209 1536Contig 210 1537 Contig 211 1538 Contig 212 1539 Contig 213 1540 Contig214 1541 Contig 215 1542 Contig 216 1543 Contig 217 1544 Contig 218 1545Contig 219 1546 Contig 220 1547 Contig 221 1548 Contig 222 1549 Contig223 1550 Contig 224 1551 Contig 225 1552 Contig 226 1553 Contig 227 1554Contig 228 1555 Contig 229 1556 Contig 230 1557 Contig 231 1558 Contig232 1559 Contig 233 1560 Contig 234 1561 Contig 235 1562 Contig 236 1563Contig 237

TABLE 7 SEQ ID CLONE NO IDENTIFIER 1564 R0124:E05 1565 R0124:E06 1566R0124:E08 1567 R0124:F07 1568 R0124:F08 1569 R0124:F09 1570 R0124:G041571 R0129:A02 1572 R0129:A03 1573 R0129:A06 1574 R0129:A07 1575R0129:A08 1576 R0129:A09 1577 R0129:A10 1578 R0129:A11 1579 R0129:A121580 R0129:B02 1581 R0129:B03 1582 R0129:B04 1583 R0129:B05 1584R0129:B06 1585 R0129:B07 1586 R0129:B08 1587 R0129:B09 1588 R0129:B101589 R0129:B11 1590 R0129:B12 1591 R0129:C01 1592 R0129:C02 1593R0129:C03 1594 R0129:C04 1595 R0129:C06 1596 R0129:C07 1597 R0129:C081598 R0129:C09 1599 R0129:C10 1600 R0129:C11 1601 R0129:C12 1602R0129:D01 1603 R0129:D03 1604 R0129:D04 1605 R0129:D05 1606 R0129:D061607 R0129:D07 1608 R0129:D08 1609 R0129:D09 1610 R0129:D10 1611R0129:D11 1612 R0129:E02 1613 R0129:E03 1614 R0129:E04 1615 R0129:E051616 R0129:E06 1617 R0129:E07 1618 R0129:E08 1619 R0129:E09 1620R0129:E11 1621 R0129:E12 1622 R0129:F01 1623 R0129:F02 1624 R0129:F031625 R0129:F04 1626 R0129:F06 1627 R0129:F07 1628 R0129:F08 1629R0129:F09 1630 R0129:F10 1631 R0129:F11 1632 R0129:F12 1633 R0129:G011634 R0129:G02 1635 R0129:G03 1636 R0129:G04 1637 R0129:G05 1638R0129:G06 1639 R0129:G07 1640 R0129:G08 1641 R0129:G09 1642 R0129:G101643 R0129:G11 1644 R0129:G12 1645 R0129:H01 1646 R0129:H02 1647R0129:H03 1648 R0129:H04 1649 R0129:H05 1650 R0129:H08 1651 R0129:H091652 R0129:H10 1653 R0129:H11

TABLE 8 SEQ ID CLONE NO IDENTIFIER 1654 26484 1655 26496 1656 26517 165726531 1658 26022 1659 26026 1660 26810 1661 26815 1662 26869 1663 268831664 26902

SEQ ID NO:1667 is the protein sequence of expressed recombinant L7548S.

SEQ ID NO:1668 is the cDNA sequence of expressed recombinant L7548S.

SEQ ID NO: 1669 is the extended cDNA sequence of clone #18971 (L801P).

SEQ ID NO: 1670 is the amino acid sequence of open reading frame ORF4encoded by SEQ ID NO:1669.

SEQ ID NO:1671 is the amino acid sequence of open reading frame ORF5encoded by SEQ ID NO:1669.

SEQ ID NO: 1672 is the amino acid sequence of open reading frame ORF6encoded by SEQ ID NO:1669.

SEQ ID NO: 1673 is the amino acid sequence of open reading frame ORF7encoded by SEQ ID NO:1669.

SEQ ID NO:1674 is the amino acid sequence of open reading frame ORF8encoded by SEQ ID NO:1669.

SEQ. ID NO: 1675 is the amino acid sequence of open reading frame ORF9encoded by SEQ ID NO:1669.

SEQ ID NO:1676 is the extended cDNA for contig 139 (SEQ ID NO:1467),also known as L985P.

SEQ ID NO:1677 is the L985P amino acid sequence encoded by SEQ ID NO:1676.

SEQ ID NO: 1678 is the amino acid sequence of open reading frame SEQ IDNO:1669.

SEQ ID NO: 1679 is the amino acid sequence of an open reading frame for(SEQ ID NO: 1467).

SEQ ID NOs: 1680-1788, set forth in the table below, represent cDNAclones identified by microarray analysis of the SQL1, SCL1, SCL3 andSCL4 libraries on lug chip 5.

SEQ ID Clone NO: Identifier 1680 58456 1681 58458 1682 58462 1683 584691684 58470 1685 58482 1686 58485 1687 58501 1688 58502 1689 58505 169058507 1691 58509 1692 58512 1693 58527 1694 58529 1695 58531 1696 585371697 58539 1698 58545 1699 59319 1700 59322 1701 59348 1702 59350 170359363 1704 59365 1705 59370 1706 59373 1707 59376 1708 61050 1709 610511710 61052 1711 61054 1712 61056 1713 61057 1714 61060 1715 61062 171661063 1717 61064 1718 61065 1719 61066 1720 61069 1721 61070 1722 610711723 61074 1724 61075 1725 61077 1726 61079 1727 61080 1728 61081 172961083 1730 61085 1731 61086 1732 61088 1733 61090 1734 61091 1735 610931736 61094 1737 61096 1738 61097 1739 61099 1740 61100 1741 61103 174261105 1743 61106 1744 61110 1745 61113 1746 61115 1747 61117 1748 611181749 61119 1750 61120 1751 61122 1752 61125 1753 61126 1754 61130 175561133 1756 61134 1757 61135 1758 61137 1759 61139 1760 61143 1761 611441762 61148 1763 61151 1764 61155 1765 61156 1766 61159 1767 61160 176861163 1769 61167 1770 61172 1771 61173 1772 61176 1773 61177 1774 611831775 61185 1776 61188 1777 61192 1778 61198 1779 61201 1780 61202 178161204 1782 61206 1783 61210 1784 61212 1785 61216 1786 61225 1787 612261788 61227

SEQ ID NO: 1789 is the cDNA sequence of clone #47988 (L972P).

SEQ ID NO: 1790 is the cDNA sequence of clone #48005 (L979P).

SEQ ID NO: 1791 is an extended cDNA sequence for clone #48005 (L979P).

SEQ ID NO: 1792 is an extended cDNA sequence for clone #49826 (SEQ IDNO: 79; L980P).

SEQ ID NO: 1793 is an extended cDNA sequence for clone #20631 (SEQ IDNO: 117; L973P).

SEQ ID NO: 1794 is an extended cDNA sequence for clone #20661 (SEQ IDNO:128; L974P).

SEQ ID NO:1795 is an extended cDNA sequence for clone #50430 (SEQ IDNO:1442; L996P).

SEQ ID NO:1796 is an extended cDNA sequence for clone #26961 (SEQ IDNO:288; L977P).

SEQ ID NO:1797 is an extended cDNA sequence for clone #24928 (SEQ ID NO:1339; L978P).

SEQ ID NO:1798 is an extended cDNA sequence for clone #50507 (SEQ IDNO:1446; L984P).

SEQ ID NO:1799 is an extended cDNA sequence for clone #50645 (SEQ IDNO:1531; L988P).

SEQ ID NO: 1800 is an extended cDNA sequence for clone #50628 (SEQ IDNO:1533; L1423P).

SEQ ID NO: 1801 is an extended cDNA sequence for clone #50560 (SEQ IDNO: 1527; L987P).

SEQ ID NO: 1802 is an extended cDNA sequence for clone #27699 (SEQ IDNO:468; L998P).

SEQ ID NO:1803 is an extended cDNA sequence for clone #59303 (SEQ IDNO:949; L1425P).

SEQ ID NO: 1804 is an extended cDNA sequence for clone #59314 (SEQ IDNO: 1156; L1426P).

SEQ ID NO: 1805 is an extended cDNA sequence for clone #59298 (SEQ IDNO:921; L1427P).

SEQ ID NO: 1806 is an amino acid sequence encoded by SEQ ID NO:1791.

SEQ ID NO: 1807 is an amino acid sequence encoded by SEQ ID NO: 1792.

SEQ ID NO: 1808 is an amino acid sequence encoded by SEQ ID NO:1793.

SEQ ID NO: 1809 is an amino acid sequence encoded by SEQ ID NO:1794.

SEQ ID NO: 1810 is an amino acid sequence encoded by SEQ ID NO:1795.

SEQ ID NO: 1811 is an amino acid sequence encoded by SEQ ID NO:1796.

SEQ ID NO: 1812 is an amino acid sequence encoded by SEQ ID NO:1797.

SEQ ID NO: 1813 is an amino acid sequence encoded by SEQ ID NO:1798.

SEQ ID NO: 1814 is an amino acid sequence encoded by SEQ ID NO:1799.

SEQ ID NO: 1815 is an amino acid sequence encoded by SEQ ID NO:1800.

SEQ ID NO: 1816 is an amino acid sequence encoded by SEQ ID NO: 1527(L987P).

SEQ ID NO: 1817 is an amino acid sequence encoded by SEQ ID NO:1823.

SEQ ID NO: 1818 is an amino acid sequence encoded by SEQ ID NO:1801.

SEQ ID NO: 1819 is an amino acid sequence encoded by SEQ ID NO:1802.

SEQ ID NO: 1820 is an amino acid sequence encoded by SEQ ID NO: 1803.

SEQ ID NO: 1821 is an amino acid sequence encoded by SEQ ID NO: 1804.

SEQ ID NO: 1822 is an amino acid sequence encoded by SEQ ID NO:1805.

SEQ ID NO: 1823 is an extended cDNA sequence for clone #50560 (SEQ IDNO: 1527; L987P).

SEQ ID NO: 1824 is an extended, full length cDNA sequence for cloneL872P (SEQ ID NO:34).

SEQ ID NO: 1825 is the amino acid sequence encoded by SEQ ID NO:1824.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed tocompositions and methods for using the compositions, for example in thetherapy and diagnosis of cancer, such as lung cancer. Certainillustrative compositions described herein include lung tumorpolypeptides, polynucleotides encoding such polypeptides, binding agentssuch as antibodies, antigen presenting cells (APCs) and/or immune systemcells (e.g., T cells). A “lung tumor protein,” as the term is usedherein, refers generally to a protein that is expressed in lung tumorcells at a level that is at least two fold, and preferably at least fivefold, greater than the level of expression in a normal tissue, asdetermined using a representative assay provided herein. Certain lungtumor proteins are tumor proteins that react detectably (within animmunoassay, such as an ELISA or Western blot) with antisera of apatient afflicted with lung cancer.

Therefore, in accordance with the above, and as described further below,the present invention provides illustrative polynucleotide compositionshaving sequences set forth in SEQ ID NO: 1-323, 341-782, 784-785, 788,790, 792, 794, 796, 800-804, 807, 808, 810-826, 878-1664, 1668, 1669,1676, 1680-1805, and 1824 illustrative polypeptide compositions havingamino acid sequences set forth in SEQ ID NO: 324-340, 783, 786, 787,789, 791, 793, 795, 797-799, 805, 806, 809, 827, 1667, 1670-1675,1677-1679, 1806-1822 and 1825 antibody compositions capable of bindingsuch polypeptides, and numerous additional embodiments employing suchcompositions, for example in the detection, diagnosis and/or therapy ofhuman lung cancer.

POLYNUCLEOTIDE COMPOSITIONS

As used herein, the terms “DNA segment” and “polynucleotide” refer to aDNA molecule that has been isolated free of total genomic DNA of aparticular species. Therefore, a DNA segment encoding a polypeptiderefers to a DNA segment that contains one or more coding sequences yetis substantially isolated away from, or purified free from, totalgenomic DNA of the species from which the DNA segment is obtained.Included within the terms “DNA segment” and “polynucleotide” are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phagemids, phage,viruses, and the like.

As will be understood by those skilled in the art, the DNA segments ofthis invention can include genomic sequences, extra-genomic andplasmid-encoded sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides, peptidesand the like. Such segments may be naturally isolated, or modifiedsynthetically by the hand of man.

“Isolated,” as used herein, means that a polynucleotide is substantiallyaway from other coding sequences, and that the DNA segment does notcontain large portions of unrelated coding DNA, such as largechromosomal fragments or other functional genes or polypeptide codingregions. Of course, this refers to the DNA segment as originallyisolated, and does not exclude genes or coding regions later added tothe segment by the hand of man.

As will be recognized by the skilled artisan, polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. RNA molecules includeHnRNA molecules, which contain introns and correspond to a DNA moleculein a one-to-one manner, and mRNA molecules, which do not containintrons. Additional coding or non-coding sequences may, but need not, bepresent within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a lung tumor protein or a portion thereof) or maycomprise a variant, or a biological or antigenic functional equivalentof such a sequence. Polynucleotide variants may contain one or moresubstitutions, additions, deletions and/or insertions, as furtherdescribed below, preferably such that the immunogenicity of the encodedpolypeptide is not diminished, relative to a native tumor protein. Theeffect on the immunogenicity of the encoded polypeptide may generally beassessed as described herein. The term “variants” also encompasseshomologous genes of xenogenic origin.

When comparing polynucleotide or polypeptide sequences, two sequencesare said to be “identical” if the sequence of nucleotides or amino acidsin the two sequences is the same when aligned for maximumcorrespondence, as described below. Comparisons between two sequencesare typically performed by comparing the sequences over a comparisonwindow to identify and compare local regions of sequence similarity. A“comparison window” as used herein, refers to a segment of at leastabout 20 contiguous positions, usually 30 to about 75, 40 to about 50,in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides and polypeptides of the invention.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. In one illustrativeexample, cumulative scores can be calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix can be used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, andexpectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff andHenikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of50, expectation (E) of 10, M=5, N=−4 and a comparison of both strands.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e., the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Therefore, the present invention encompasses polynucleotide andpolypeptide sequences having substantial identity to the sequencesdisclosed herein, for example those comprising at least 50% sequenceidentity, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to apolynucleotide or polypeptide sequence of this invention using themethods described herein, (e.g., BLAST analysis using standardparameters, as described below). One skilled in this art will recognizethat these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like.

In additional embodiments, the present invention provides isolatedpolynucleotides and polypeptides comprising various lengths ofcontiguous stretches of sequence identical to or complementary to one ormore of the sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise at least about 15, 20, 30, 40,50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguousnucleotides of one or more of the sequences disclosed herein as well asall intermediate lengths there between. It will be readily understoodthat “intermediate lengths”, in this context, means any length betweenthe quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30,31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151,152, 153, etc.; including all integers through 200-500; 500-1,000, andthe like.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, illustrative DNA segments withtotal lengths of about 10,000, about 5000, about 3000, about 2,000,about 1,000, about 500, about 200, about 100, about 50 base pairs inlength, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

In other embodiments, the present invention is directed topolynucleotides that are capable of hybridizing under moderatelystringent conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5 X SSC, 0.5% SDS,1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-65° C., 5 X SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2X, 0.5Xand 0.2X SSC containing 0.1% SDS.

Moreover, it will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention. Further, alleles of the genes comprising thepolynucleotide sequences provided herein are within the scope of thepresent invention. Alleles are endogenous genes that are altered as aresult of one or more mutations, such as deletions, additions and/orsubstitutions of nucleotides. The resulting mRNA and protein may, butneed not, have an altered structure or function. Alleles may beidentified using standard techniques (such as hybridization,amplification and/or database sequence comparison).

PROBES AND PRIMERS

In other embodiments of the present invention, the polynucleotidesequences provided herein can be advantageously used as probes orprimers for nucleic acid hybridization. As such, it is contemplated thatnucleic acid segments that comprise a sequence region of at least about15 nucleotide long contiguous sequence that has the same sequence as, oris complementary to, a 15 nucleotide long contiguous sequence disclosedherein will find particular utility. Longer contiguous identical orcomplementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200,500, 1000 (including all intermediate lengths) and even up to fulllength sequences will also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize to asequence of interest will enable them to be of use in detecting thepresence of complementary sequences in a given sample. However, otheruses are also envisioned, such as the use of the sequence informationfor the preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

Polynucleotide molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so (including intermediate lengths as well),identical or complementary to a polynucleotide sequence disclosedherein, are particularly contemplated as hybridization probes for usein, e.g., Southern and Northern blotting. This would allow a geneproduct, or fragment thereof, to be analyzed, both in diverse cell typesand also in various bacterial cells. The total size of fragment, as wellas the size of the complementary stretch(es), will ultimately depend onthe intended use or application of the particular nucleic acid segment.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 15 and about 100 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

The use of a hybridization probe of about 15-25 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 15 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 25 contiguous nucleotides,or even longer where desired.

Hybridization probes may be selected from any portion of any of thesequences disclosed herein. All that is required is to review a sequenceset forth herein or to any continuous portion of the sequence, fromabout 15-25 nucleotides in length up to and including the full lengthsequence, that one wishes to utilize as a probe or primer. The choice ofprobe and primer sequences may be governed by various factors. Forexample, one may wish to employ primers from towards the termini of thetotal sequence.

Small polynucleotide segments or fragments may be readily prepared by,for example, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U. S. Pat. No. 4,683,202(incorporated herein by reference), by introducing selected sequencesinto recombinant vectors for recombinant production, and by otherrecombinant DNA techniques generally known to those of skill in the artof molecular biology.

The nucleotide sequences of the invention may be used for their abilityto selectively form duplex molecules with complementary stretches of theentire gene or gene fragments of interest. Depending on the applicationenvisioned, one will typically desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by a salt concentration of fromabout 0.02 M to about 0.15 M salt at temperatures of from about 50° C.to about 70° C. Such selective conditions tolerate little, if any,mismatch between the probe and the template or target strand, and wouldbe particularly suitable for isolating related sequences.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template, less stringent (reduced stringency) hybridizationconditions will typically be needed in order to allow formation of theheteroduplex. In these circumstances, one may desire to employ saltconditions such as those of from about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Cross-hybridizingspecies can thereby be readily identified as positively hybridizingsignals with respect to control hybridizations. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

POLYNUCLEOTIDE IDENTIFICATION AND CHARACTERIZATION

Polynucleotides may be identified, prepared and/or manipulated using anyof a variety of well established techniques. For example, apolynucleotide may be identified, as described in more detail below, byscreening a microarray of cDNAs for tumor-associated expression (i.e.,expression that is at least two fold greater in a tumor than in normaltissue, as determined using a representative assay provided herein).Such screens may be performed, for example, using a Synteni microarray(Palo Alto, Calif.) according to the manufacturer's instructions (andessentially as described by Schena et al., Proc. Natl. Acad. Sci. USA93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA94:2150-2155, 1997). Alternatively, polynucleotides may be amplifiedfrom cDNA prepared from cells expressing the proteins described herein,such as lung tumor cells. Such polynucleotides may be amplified viapolymerase chain reaction (PCR). For this approach, sequence-specificprimers may be designed based on the sequences provided herein, and maybe purchased or synthesized.

An amplified portion of a polynucleotide of the present invention may beused to isolate a full length gene from a suitable library (e.g., a lungtumor cDNA library) using well known techniques. Within such techniques,a library (cDNA or genomic) is screened using one or more polynucleotideprobes or primers suitable for amplification. Preferably, a library issize-selected to include larger molecules. Random primed libraries mayalso be preferred for identifying 5′ and upstream regions of genes.Genomic libraries are preferred for obtaining introns and extending 5′sequences.

For hybridization techniques, a partial sequence may be labeled (e.g.,by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. cDNA clones may be analyzed to determine the amount ofadditional sequence by, for example, PCR using a primer from the partialsequence and a primer from the vector. Restriction maps and partialsequences may be generated to identify one or more overlapping clones.The complete sequence may then be determined using standard techniques,which may involve generating a series of deletion clones. The resultingoverlapping sequences can then assembled into a single contiguoussequence. A full length cDNA molecule can be generated by ligatingsuitable fragments, using well known techniques.

Alternatively, there are numerous amplification techniques for obtaininga full length coding sequence from a partial cDNA sequence. Within suchtechniques, amplification is generally performed via PCR. Any of avariety of commercially available kits may be used to perform theamplification step. Primers may be designed using, for example, softwarewell known in the art. Primers are preferably 22-30 nucleotides inlength, have a GC content of at least 50% and anneal to the targetsequence at temperatures of about 68° C. to 72° C. The amplified regionmay be sequenced as described above, and overlapping sequences assembledinto a contiguous sequence.

One such amplification technique is inverse PCR (see Triglia et al.,Nucl. Acids Res. 16:8186, 1988), which uses restriction enzymes togenerate a fragment in the known region of the gene. The fragment isthen circularized by intramolecular ligation and used as a template forPCR with divergent primers derived from the known region. Within analternative approach, sequences adjacent to a partial sequence may beretrieved by amplification with a primer to a linker sequence and aprimer specific to a known region. The amplified sequences are typicallysubjected to a second round of amplification with the same linker primerand a second primer specific to the known region. A variation on thisprocedure, which employs two primers that initiate extension in oppositedirections from the known sequence, is described in WO 96/38591. Anothersuch technique is known as “rapid amplification of cDNA ends” or RACE.This technique involves the use of an internal primer and an externalprimer, which hybridizes to a polyA region or vector sequence, toidentify sequences that are 5′ and 3′ of a known sequence. Additionaltechniques include capture PCR (Lagerstrom et al., PCR Methods Applic.1:111-19, 1991) and walking PCR (Parker et al., Nucl. Acids. Res.19:3055-60, 1991). Other methods employing amplification may also beemployed to obtain a full length cDNA sequence.

In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

POLYNUCLEOTIDE EXPRESSION IN HOST CELLS

In other embodiments of the invention, polynucleotide sequences orfragments thereof which encode polypeptides of the invention, or fusionproteins or functional equivalents thereof, may be used in recombinantDNA molecules to direct expression of a polypeptide in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other DNAsequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and these sequences maybe used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. For example, DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

Sequences encoding a desired polypeptide may be synthesized, in whole orin part, using chemical methods well known in the art (see Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al.(1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the proteinitself may be produced using chemical methods to synthesize the aminoacid sequence of a polypeptide, or a portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(Roberge, J. Y. et al. (1995) Science 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431 A PeptideSynthesizer (Perkin Elmer, Palo Alto, Calif.).

A newly synthesized peptide may be substantially purified by preparativehigh performance liquid chromatography (e.g., Creighton, T. (1983)Proteins, Structures and Molecular Principles, WH Freeman and Co., NewYork, N.Y.) or other comparable techniques available in the art. Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure).Additionally, the amino acid sequence of a polypeptide, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

In order to express a desired polypeptide, the nucleotide sequencesencoding the polypeptide, or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook, J. et al.(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York. N.Y.

A variety of expression vector/host systems may be utilized to containand express polynucleotide sequences. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are needed, for example for the inductionof antibodies, vectors which direct high level expression of fusionproteins that are readily purified may be used. Such vectors include,but are not limited to, the multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of .beta.-galactosidase so that a hybrid proteinis produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega,Madison, Wis.) may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhard,E. K. et al. (1994) Proc. Natl. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc.Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation.glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) geneswhich can be employed in tk.sup.- or aprt.sup.- cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, beta-glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA—DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include membrane, solution, or chipbased technologies for the detection and/or quantification of nucleicacid or protein.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on a given polypeptide may be preferred forsome applications, but a competitive binding assay may also be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul. Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3:263-281) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.12:441-453).

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield J. (1963) J. Am.Chem. Soc. 85:2149-2154). Protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431 A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

SITE-SPECIFIC MUTAGENESIS

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent polypeptides,through specific mutagenesis of the underlying polynucleotides thatencode them. The technique, well-known to those of skill in the art,further provides a ready ability to prepare and test sequence variants,for example, incorporating one or more of the foregoing considerations,by introducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments of the present invention, the inventorscontemplate the mutagenesis of the disclosed polynucleotide sequences toalter one or more properties of the encoded polypeptide, such as theantigenicity of a polypeptide vaccine. The techniques of site-specificmutagenesis are well-known in the art, and are widely used to createvariants of both polypeptides and polynucleotides. For example,site-specific mutagenesis is often used to alter a specific portion of aDNA molecule. In such embodiments, a primer comprising typically about14 to about 25 nucleotides or so in length is employed, with about 5 toabout 10 residues on both sides of the junction of the sequence beingaltered.

As will be appreciated by those of skill in the art, site-specificmutagenesis techniques have often employed a phage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage are readily commercially-available and their use isgenerally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982, each incorporated herein byreference, for that purpose.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U. S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

POLYNUCLEOTIDE AMPLIFICATION TECHNIQUES

A number of template dependent processes are available to amplify thetarget sequences of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR™) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, each of which is incorporated herein by reference in itsentirety. Briefly, in PCR™, two primer sequences are prepared which arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase (e.g., Taq polymerase). Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe target sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction product and the process isrepeated. Preferably reverse transcription and PCR™ amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

Another method for amplification is the ligase chain reaction (referredto as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308 (specificallyincorporated herein by reference in its entirety). In LCR, twocomplementary probe pairs are prepared, and in the presence of thetarget sequence, each pair will bind to opposite complementary strandsof the target such that they abut. In the presence of a ligase, the twoprobe pairs will link to form a single unit. By temperature cycling, asin PCR™, bound ligated units dissociate from the target and then serveas “target sequences” for ligation of excess probe pairs. U.S. Pat. No.4,883,750, incorporated herein by reference in its entirety, describesan alternative method of amplification similar to LCR for binding probepairs to a target sequence.

Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No.PCT/US87/00880, incorporated herein by reference in its entirety, mayalso be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA that has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[α-thio]triphosphates in one strand of arestriction site (Walker et al., 1992, incorporated herein by referencein its entirety), may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e. nick translation. Asimilar method, called Repair Chain Reaction (RCR) is another method ofamplification which may be useful in the present invention and isinvolves annealing several probes throughout a region targeted foramplification, followed by a repair reaction in which only two of thefour bases are present. The other two bases can be added as biotinylatedderivatives for easy detection. A similar approach is used in SDA.

Sequences can also be detected using a cyclic probe reaction (CPR). InCPR, a probe having a 3′ and 5′ sequences of non-target DNA and aninternal or “middle” sequence of the target protein specific RNA ishybridized to DNA which is present in a sample. Upon hybridization, thereaction is treated with RNaseH, and the products of the probe areidentified as distinctive products by generating a signal that isreleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated. Thus, CPR involvesamplifying a signal generated by hybridization of a probe to a targetgene specific expressed nucleic acid.

Still other amplification methods described in Great Britain Pat. Appl.No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No. PCT/US89/01025,each of which is incorporated herein by reference in its entirety, maybe used in accordance with the present invention. In the formerapplication, “modified” primers are used in a PCR-like, template andenzyme dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes is added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS) (Kwoh et al., 1989; PCT Intl. Pat. Appl.Publ. No. WO 88/10315, incorporated herein by reference in itsentirety), including nucleic acid sequence based amplification (NASBA)and 3SR. In NASBA, the nucleic acids can be prepared for amplificationby standard phenol/chloroform extraction, heat denaturation of a sample,treatment with lysis buffer and minispin columns for isolation of DNAand RNA or guanidinium chloride extraction of RNA. These amplificationtechniques involve annealing a primer that has sequences specific to thetarget sequence. Following polymerization, DNA/RNA hybrids are digestedwith RNase H while double stranded DNA molecules are heat-denaturedagain. In either case the single stranded DNA is made fully doublestranded by addition of second target-specific primer, followed bypolymerization. The double stranded DNA molecules are then multiplytranscribed by a polymerase such as T7 or SP6. In an isothermal cyclicreaction, the RNAs are reverse transcribed into DNA, and transcribedonce again with a polymerase such as T7 or SP6. The resulting products,whether truncated or complete, indicate target-specific sequences.

Eur. Pat. Appl. Publ. No. 329,822, incorporated herein by reference inits entirety, disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from resultingDNA:RNA duplex by the action of ribonuclease H (RNase H, an RNasespecific for RNA in a duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to its template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. Coli DNA polymerase I), resulting as a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein byreference in its entirety, disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic; i.e. new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” (Frohman, 1990),and “one-sided PCR” (Ohara, 1989) which are well-known to those of skillin the art.

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide (Wu andDean, 1996, incorporated herein by reference in its entirety), may alsobe used in the amplification of DNA sequences of the present invention.

BIOLOGICAL FUNCTIONAL EQUIVALENTS

Modification and changes may be made in the structure of thepolynucleotides and polypeptides of the present invention and stillobtain a functional molecule that encodes a polypeptide with desirablecharacteristics. As mentioned above, it is often desirable to introduceone or more mutations into a specific polynucleotide sequence. Incertain circumstances, the resulting encoded polypeptide sequence isaltered by this mutation, or in other cases, the sequence of thepolypeptide is unchanged by one or more mutations in the encodingpolynucleotide.

When it is desirable to alter the amino acid sequence of a polypeptideto create an equivalent, or even an improved, second-generationmolecule, the amino acid changes may be achieved by changing one or moreof the codons of the encoding DNA sequence, according to Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can,. be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss-of their biological utility or activity.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within +1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(specifically incorporated herein by reference in its entirety), statesthat the greatest local average hydrophilicity of a protein, as governedby the hydrophilicity of its adjacent amino acids, correlates with abiological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within +1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

In addition, any polynucleotide may be further modified to increasestability in vivo. Possible modifications include, but are not limitedto, the addition of flanking sequences at the 5′ and/or 3′ ends; the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine and wybutosine, as well as acetyl- methyl-,thio- and other modified forms of adenine, cytidine, guanine, thymineand uridine.

IN VIVO POLYNUCLEOTIDE DELIVERY TECHNIQUES

In additional embodiments, genetic constructs comprising one or more ofthe polynucleotides of the invention are introduced into cells in vivo.This may be achieved using any of a variety or well known approaches,several of which are outlined below for the purpose of illustration.

1. ADENOVIRUS

One of the preferred methods for in vivo delivery of one or more nucleicacid sequences involves the use of an adenovirus expression vector.“Adenovirus expression vector” is meant to include those constructscontaining adenovirus sequences sufficient to (a) support packaging ofthe construct and (b) to express a polynucleotide that has been clonedtherein in a sense or antisense orientation. Of course, in the contextof an antisense construct, expression does not require that the geneproduct be synthesized.

The expression vector comprises a genetically engineered form of anadenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNA's issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them preferredmRNA's for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was ransformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kB of DNA. Combined with theapproximately 5.5 kB of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kB, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1-deleted virus is incomplete. For example, leakage of viral geneexpression has been observed with the currently available vectors athigh multiplicities of infection (MOI) (Mulligan, 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the currently preferred helper cell line is 293.

Recently, Racher et al. (1995) disclosed improved methods for culturing293 cells and propagating adenovirus. In one format, natural cellaggregates are grown by inoculating individual cells into 1 litersiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcarriers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and left stationary, with occasional agitation, for1 to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain aconditional replication-defective adenovirus vector for use in thepresent invention, since Adenovirus type 5 is a human adenovirus aboutwhich a great deal of biochemical and genetic information is known, andit has historically been used for most constructions employingadenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed by Karlsson et al. (1986) or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹-10¹¹ plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

2. RETROVIRUSES

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding oneor more oligonucleotide or polynucleotide sequences of interest isinserted into the viral genome in the place of certain viral sequencesto produce a virus that is replication-defective. In order to producevirions, a packaging cell line containing the gag, pol, and env genesbut without the LTR and packaging components is constructed (Mann etal., 1983). When a recombinant plasmid containing a cDNA, together withthe retroviral LTR and packaging sequences is introduced into this cellline (by calcium phosphate precipitation for example), the packagingsequence allows the RNA transcript of the recombinant plasmid to bepackaged into viral particles, which are then secreted into the culturemedia (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983).The media containing the recombinant retroviruses is then collected,optionally concentrated, and used for gene transfer. Retroviral vectorsare able to infect a broad variety of cell types. However, integrationand stable expression require the division of host cells (Paskind etal., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

3. ADENO-ASSOCIATED VIRUSES

AAV (Ridgeway, 1988; Hermonat and Muzycska, 1984) is a parovirus,discovered as a contamination of adenoviral stocks. It is a ubiquitousvirus (antibodies are present in 85% of the US human population) thathas not been linked to any disease. It is also classified as adependovirus, because its replications is dependent on the presence of ahelper virus, such as adenovirus. Five serotypes have been isolated, ofwhich AAV-2 is the best characterized. AAV has a single-stranded linearDNA that is encapsidated into capsid proteins VP1, VP2 and VP3 to forman icosahedral virion of 20 to 24 nm in diameter (Muzyczka andMcLaughlin, 1988).

The AAV DNA is approximately 4.7 kilobases long. It contains two openreading frames and is flanked by two ITRs. There are two major genes inthe AAV genome: rep and cap. The rep gene codes for proteins responsiblefor viral replications, whereas cap codes for capsid protein VP1-3. EachITR forms a T-shaped hairpin structure. These terminal repeats are theonly essential cis components of the AAV for chromosomal integration.Therefore, the AAV can be used as a vector with all viral codingsequences removed and replaced by the cassette of genes for delivery.Three viral promoters have been identified and named p5, p19, and p40,according to their map position. Transcription from p5 and p19 resultsin production of rep proteins, and transcription from p40 produces thecapsid proteins (Hermonat and Muzyczka, 1984).

There are several factors that prompted researchers to study thepossibility of using rAAV as an expression vector One is that therequirements for delivering a gene to integrate into the host chromosomeare surprisingly few. It is necessary to have the 145-bp ITRs, which areonly 6% of the AAV genome. This leaves room in the vector to assemble a4.5-kb DNA insertion. While this carrying capacity may prevent the AAVfrom delivering large genes, it is amply suited for delivering theantisense constructs of the present invention.

AAV is also a good choice of delivery vehicles due to its safety. Thereis a relatively complicated rescue mechanism: not only wild typeadenovirus but also AAV genes are required to mobilize rAAV. Likewise,AAV is not pathogenic and not associated with any disease. The removalof viral coding sequences minimizes immune reactions to viral geneexpression, and therefore, rAAV does not evoke an inflammatory response.

4. OTHER VIRAL VECTORS AS EXPRESSION CONSTRUCTS

Other viral vectors may be employed as expression constructs in thepresent invention for the delivery of oligonucleotide or polynucleotidesequences to a host cell. Vectors derived from viruses such as vacciniavirus (Ridgeway, 1988; Coupar et al., 1988), lentiviruses, polio virusesand herpes viruses may be employed. They offer several attractivefeatures for various mammalian cells (Friedmann, 1989; Ridgeway, 1988;Coupar et al., 1988; Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. (1991) introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al., 1991).

5. NON-VIRAL VECTORS

In order to effect expression of the oligonucleotide or polynucleotidesequences of the present invention, the expression construct must bedelivered into a cell. This delivery may be accomplished in vitro, as inlaboratory procedures for transforming cells lines, or in vivo or exvivo, as in the treatment of certain disease states. As described above,one preferred mechanism for delivery is via viral infection where theexpression construct is encapsulated in an infectious viral particle.

Once the expression construct has been delivered into the cell thenucleic acid encoding the desired oligonucleotide or polynucleotidesequences may be positioned and expressed at different sites. In certainembodiments, the nucleic acid encoding the construct may be stablyintegrated into the genome of the cell. This integration may be in thespecific location and orientation via homologous recombination (genereplacement) or it may be integrated in a random, non-specific location(gene augmentation). In yet further embodiments, the nucleic acid may bestably maintained in the cell as a separate, episomal segment of DNA.Such nucleic acid segments or “episomes” encode sequences sufficient topermit maintenance and replication independent of or in synchronizationwith the host cell cycle. How the expression construct is delivered to acell and where in the cell the nucleic acid remains is dependent on thetype of expression construct employed.

In certain embodiments of the invention, the expression constructcomprising one or more oligonucleotide or polynucleotide sequences maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Reshef (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e. ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

ANTISENSE OLIGONUCLEOTIDES

The end result of the flow of genetic information is the synthesis ofprotein. DNA is transcribed by polymerases into messenger RNA andtranslated on the ribosome to yield a folded, functional protein. Thusthere are several steps along the route where protein synthesis can beinhibited. The native DNA segment coding for a polypeptide describedherein, as all such mammalian DNA strands, has two strands: a sensestrand and an antisense strand held together by hydrogen bonding. Themessenger RNA coding for polypeptide has the same nucleotide sequence asthe sense DNA strand except that the DNA thymidine is replaced byuridine. Thus, synthetic antisense nucleotide sequences will bind to amRNA and inhibit expression of the protein encoded by that mRNA.

The targeting of antisense oligonucleotides to mRNA is thus onemechanism to shut down protein synthesis, and, consequently, representsa powerful and targeted therapeutic approach. For example, the synthesisof polygalactauronase and the muscarine type 2 acetylcholine receptorare inhibited by antisense oligonucleotides directed to their respectivemRNA sequences (U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829,each specifically incorporated herein by reference in its entirety).Further, examples of antisense inhibition have been demonstrated withthe nuclear protein cyclin, the multiple drug resistance gene (MDG1),ICAM-1, E-selectin, STK-1, striatal GABA_(A) receptor and human EGF(Jaskulski et al., 1988; Vasanthakumar and Ahmed, 1989; Peris et al.,1998; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573; U.S. Pat. No.5,718,709 and U.S. Pat. No. 5,610,288, each specifically incorporatedherein by reference in its entirety). Antisense constructs have alsobeen described that inhibit and can be used to treat a variety ofabnormal cellular proliferations, e.g. cancer (U.S. Pat. No. 5,747,470;U.S. Pat. No. 5,591,317 and U.S. Pat. No. 5,783,683, each specificallyincorporated herein by reference in its entirety).

Therefore, in exemplary embodiments, the invention providesoligonucleotide sequences that comprise all, or a portion of, anysequence that is capable of specifically binding to polynucleotidesequence described herein, or a complement thereof. In one embodiment,the antisense oligonucleotides comprise DNA or derivatives thereof. Inanother embodiment, the oligonucleotides comprise RNA or derivativesthereof. In a third embodiment, the oligonucleotides are modified DNAscomprising a phosphorothioated modified backbone. In a fourthembodiment, the oligonucleotide sequences comprise peptide nucleic acidsor derivatives thereof. In each case, preferred compositions comprise asequence region that is complementary, and more preferablysubstantially-complementary, and even more preferably, completelycomplementary to one or more portions of polynucleotides disclosedherein.

Selection of antisense compositions specific for a given gene sequenceis based upon analysis of the chosen target sequence (i.e. in theseillustrative examples the rat and human sequences) and determination ofsecondary structure, T_(m), binding energy, relative stability, andantisense compositions were selected based upon their relative inabilityto form dimers, hairpins, or other secondary structures that wouldreduce or prohibit specific binding to the target mRNA in a host cell.

Highly preferred target regions of the mRNA, are those which are at ornear the AUG translation initiation codon, and those sequences whichwere substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationswere performed using v.4 of the OLIGO primer analysis software (Rychlik,1997) and the BLASTN 2.0.5 algorithm software (Altschul et al., 1997).

The use of an antisense delivery method employing a short peptidevector, termed MPG (27 residues), is also contemplated. The MPG peptidecontains a hydrophobic domain derived from the fusion sequence of HIVgp41 and a hydrophilic domain from the nuclear localization sequence ofSV40 T-antigen (Morris et al., 1997). It has been demonstrated thatseveral molecules of the MPG peptide coat the antisense oligonucleotidesand can be delivered into cultured mammalian cells in less than 1 hourwith relatively high efficiency (90%). Further, the interaction with MPGstrongly increases both the stability of the oligonucleotide to nucleaseand the ability to cross the plasma membrane (Morris et al., 1997).

RIBOZYMES

Although proteins traditionally have been used for catalysis of nucleicacids, another class of macromolecules has emerged as useful in thisendeavor. Ribozymes are RNA-protein complexes that cleave nucleic acidsin a site-specific fashion. Ribozymes have specific catalytic domainsthat possess endonuclease activity (Kim and Cech, 1987; Gerlach et al.,1987; Forster and Symons, 1987). For example, a large number ofribozymes accelerate phosphoester transfer reactions with a high degreeof specificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855(specifically incorporated herein by reference) reports that certainribozymes can act as endonucleases with a sequence specificity greaterthan that of known ribonucleases and approaching that of the DNArestriction enzymes. Thus, sequence-specific ribozyme-mediatedinhibition of gene expression may be particularly suited to therapeuticapplications (Scanlon et al., 1991; Sarver et al., 1990). Recently, itwas reported that ribozymes elicited genetic changes in some cells linesto which they were applied; the altered genes included the oncogenesH-ras, c-fos and genes of HIV. Most of this work involved themodification of a target mRNA, based on a specific mutant codon that iscleaved by a specific ribozyme.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofa enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., 1992). Thus, thespecificity of action of a ribozyme is greater than that of an antisenseoligonucleotide binding the same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. (1992).Examples of hairpin motifs are described by Hampel et al. (Eur. Pat.Appl. Publ. No. EP 0360257), Hampel and Tritz (1989), Hampel et al.(1990) and U.S. Pat. No. 5,631,359 (specifically incorporated herein byreference). An example of the hepatitis δ virus motif is described byPerrotta and Been (1992); an example of the RNaseP motif is described byGuerrier-Takada et al. (1983); Neurospora VS RNA ribozyme motif isdescribed by Collins (Saville and Collins, 1990; Saville and Collins,1991; Collins and Olive, 1993); and an example of the Group I intron isdescribed in (U.S. Pat. No. 4,987,071, specifically incorporated hereinby reference). All that is important in an enzymatic nucleic acidmolecule of this invention is that it has a specific substrate bindingsite which is complementary to one or more of the target gene RNAregions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

In certain embodiments, it may be important to produce enzymaticcleaving agents which exhibit a high degree of specificity for the RNAof a desired target, such as one of the sequences disclosed herein. Theenzymatic nucleic acid molecule is preferably targeted to a highlyconserved sequence region of a target mRNA. Such enzymatic nucleic acidmolecules can be delivered exogenously to specific cells as required.Alternatively, the ribozymes can be expressed from DNA or RNA vectorsthat are delivered to specific cells.

Small enzymatic nucleic acid motifs (e.g., of the hammerhead or thehairpin structure) may also be used for exogenous delivery. The simplestructure of these molecules increases the ability of the enzymaticnucleic acid to invade targeted regions of the mRNA structure.Alternatively, catalytic RNA molecules can be expressed within cellsfrom eukaryotic promoters (e.g., Scanlon et al., 1991; Kashani-Sabet etal., 1992; Dropulic et al., 1992; Weerasinghe et al., 1991; Ojwang etal., 1992; Chen et al., 1992; Sarver et al., 1990). Those skilled in theart realize that any ribozyme can be expressed in eukaryotic cells fromthe appropriate DNA vector. The activity of such ribozymes can beaugmented by their release from the primary transcript by a secondribozyme (Int. Pat. Appl. Publ. No. WO 93/23569, and Int. Pat. Appl.Publ. No. WO 94/02595, both hereby incorporated by reference; Ohkawa etal., 1992; Taira et al., 1991; and Ventura et al., 1993).

Ribozymes may be added directly, or can be complexed with cationiclipids, lipid complexes, packaged within liposomes, or otherwisedelivered to target cells. The RNA or RNA complexes can be locallyadministered to relevant tissues ex vivo, or in vivo through injection,aerosol inhalation, infusion pump or stent, with or without theirincorporation in biopolymers.

Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specificallyincorporated herein by reference) and synthesized to be tested in vitroand in vivo, as described. Such ribozymes can also be optimized fordelivery. While specific examples are provided, those in the art willrecognize that equivalent RNA targets in other species can be utilizedwhen necessary.

Hammerhead or hairpin ribozymes may be individually analyzed by computerfolding (Jaeger et al., 1989) to assess whether the ribozyme sequencesfold into the appropriate secondary structure. Those ribozymes withunfavorable intramolecular interactions between the binding arms and thecatalytic core are eliminated from consideration. Varying binding armlengths can be chosen to optimize activity. Generally, at least 5 or sobases on each arm are able to bind to, or otherwise interact with, thetarget RNA.

Ribozymes of the hammerhead or hairpin motif may be designed to annealto various sites in the mRNA message, and can be chemically synthesized.The method of synthesis used follows the procedure for normal RNAsynthesis as described in Usman et al. (1987) and in Scaringe et al.(1990) and makes use of common nucleic acid protecting and couplinggroups, such as dimethoxytrityl at the 5′-end, and phosphoramidites atthe 3′-end. Average stepwise coupling yields are typically >98%. Hairpinribozymes may be synthesized in two parts and annealed to reconstruct anactive ribozyme (Chowrira and Burke, 1992). Ribozymes may be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see e.g., Usman and Cedergren, 1992). Ribozymes may bepurified by gel electrophoresis using general methods or by highpressure liquid chromatography and resuspended in water.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al, 1990;Pieken et al., 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ.No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes thegeneral methods for delivery of enzymatic RNA molecules. Ribozymes maybe administered to cells by a variety of methods known to those familiarto the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl.Publ. No. WO 93/23569, each specifically incorporated herein byreference.

Another means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol II or pol III promoters will be expressed at high levels in allcells; the levels of a given pol II promoter in a given cell type willdepend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersmay also be used, providing that the prokaryotic RNA polymerase enzymeis expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gaoand Huang, 1993; Lieber et al., 1993; Zhou et al., 1990). Ribozymesexpressed from such promoters can function in mammalian cells (e.g.Kashani-Saber et al., 1992; Ojwang et al., 1992; Chen et al., 1992; Yuet al., 1993; L'Huillier et al., 1992; Lisziewicz et al., 1993). Suchtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated vectors), or viral RNA vectors (such as retroviral,semliki forest virus, sindbis virus vectors).

Ribozymes may be used as diagnostic tools to examine genetic drift andmutations within diseased cells. They can also be used to assess levelsof the target RNA molecule. The close relationship between ribozymeactivity and the structure of the target RNA allows the detection ofmutations in any region of the molecule which alters the base-pairingand three-dimensional structure of the target RNA. By using multipleribozymes, one may map nucleotide changes which are important to RNAstructure and function in vitro, as well as in cells and tissues.Cleavage of target RNAs with ribozymes may be used to inhibit geneexpression and define the role (essentially) of specified gene productsin the progression of disease. In this manner, other genetic targets maybe defined as important mediators of the disease. These studies willlead to better treatment of the disease progression by affording thepossibility of combinational therapies (e.g., multiple ribozymestargeted to different genes, ribozymes coupled with known small moleculeinhibitors, or intermittent treatment with combinations of ribozymesand/or other chemical or biological molecules). Other in vitro uses ofribozymes are well known in the art, and include detection of thepresence of mRNA associated with an IL-5 related condition. Such RNA isdetected by determining the presence of a cleavage product aftertreatment with a ribozyme using standard methodology.

PEPTIDE NUCLEIC ACIDS

In certain embodiments, the inventors contemplate the use of peptidenucleic acids (PNAs) in the practice of the methods of the invention.PNA is a DNA mimic in which the nucleobases are attached to apseudopeptide backbone (Good and Nielsen, 1997). PNA is able to beutilized in a number methods that traditionally have used RNA or DNA.Often PNA sequences perform better in techniques than the correspondingRNA or DNA sequences and have utilities that are not inherent to RNA orDNA. A review of PNA including methods of making, characteristics of,and methods of using, is provided by Corey (1997) and is incorporatedherein by reference. As such, in certain embodiments, one may preparePNA sequences that are complementary to one or more portions of the ACEmRNA sequence, and such PNA compositions may be used to regulate, alter,decrease, or reduce the translation of ACE-specific mRNA, and therebyalter the level of ACE activity in a host cell to which such PNAcompositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normalphosphodiester backbone of DNA (Nielsen et al., 1991; Hanvey et al.,1992; Hyrup and Nielsen, 1996; Neilsen, 1996). This chemistry has threeimportant consequences: firstly, in contrast to DNA or phosphorothioateoligonucleotides, PNAs are neutral molecules; secondly, PNAs areachiral, which avoids the need to develop a stereoselective synthesis;and thirdly, PNA synthesis uses standard Boc (Dueholm et al., 1994) orFmoc (Thomson et al., 1995) protocols for solid-phase-peptide synthesis,although other methods, including a modified Merrifield method, havebeen used (Christensen et al., 1995).

PNA monomers or ready-made oligomers are commercially available fromPerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Bocor Fmoc protocols are straightforward using manual or automatedprotocols (Norton et al., 1995). The manual protocol lends itself to theproduction of chemically modified PNAs or the simultaneous synthesis offamilies of closely related PNAs.

As with peptide synthesis, the success of a particular PNA synthesiswill depend on the properties of the chosen sequence. For example, whilein theory PNAs can incorporate any combination of nucleotide bases, thepresence of adjacent purines can lead to deletions of one or moreresidues in the product. In expectation of this difficulty, it issuggested that, in producing PNAs with adjacent purines, one shouldrepeat the coupling of residues likely to be added inefficiently. Thisshould be followed by the purification of PNAs by reverse-phasehigh-pressure liquid chromatography (Norton et al., 1995) providingyields and purity of product similar to those observed during thesynthesis of peptides.

Modifications of PNAs for a given application may be accomplished bycoupling amino acids during solid-phase synthesis or by attachingcompounds that contain a carboxylic acid group to the exposed N-terminalamine. Alternatively, PNAs can be modified after synthesis by couplingto an introduced lysine or cysteine. The ease with which PNAs can bemodified facilitates optimization for better solubility or for specificfunctional requirements. Once synthesized, the identity of PNAs andtheir derivatives can be confirmed by mass spectrometry. Several studieshave made and utilized modifications of PNAs (Norton et al., 1995;Haaima et al., 1996; Stetsenko et al., 1996; Petersen et al., 1995;Ulmann et al., 1996; Koch et al., 1995; Orum et al., 1995; Footer etal., 1996; Griffith et al., 1995; Kremsky et al., 1996; Pardridge etal., 1995; Boffa et al., 1995; Landsdorp et al., 1996;Gambacorti-Passerini et al., 1996; Armitage et al., 1997; Seeger et al.,1997; Ruskowski et al., 1997). U.S. Pat. No. 5,700,922 discussesPNA-DNA-PNA chimeric molecules and their uses in diagnostics, modulatingprotein in organisms, and treatment of conditions susceptible totherapeutics.

In contrast to DNA and RNA, which contain negatively charged linkages,the PNA backbone is neutral. In spite of this dramatic alteration, PNAsrecognize complementary DNA and RNA by Watson-Crick pairing (Egholm etal., 1993), validating the initial modeling by Nielsen et al. (1991).PNAs lack 3′ to 5′ polarity and can bind in either parallel orantiparallel fashion, with the antiparallel mode being preferred (Egholmet al., 1993).

Hybridization of DNA oligonucleotides to DNA and RNA is destabilized byelectrostatic repulsion between the negatively charged phosphatebackbones of the complementary strands. By contrast, the absence ofcharge repulsion in PNA-DNA or PNA-RNA duplexes increases the meltingtemperature (T_(m)) and reduces the dependence of T_(m) on theconcentration of mono- or divalent cations (Nielsen et al., 1991). Theenhanced rate and affinity of hybridization are significant because theyare responsible for the surprising ability of PNAs to perform strandinvasion of complementary sequences within relaxed double-stranded DNA.In addition, the efficient hybridization at inverted repeats suggeststhat PNAs can recognize secondary structure effectively withindouble-stranded DNA. Enhanced recognition also occurs with PNAsimmobilized on surfaces, and Wang et al. have shown that support-boundPNAs can be used to detect hybridization events (Wang et al., 1996).

One might expect that tight binding of PNAs to complementary sequenceswould also increase binding to similar (but not identical) sequences,reducing the sequence specificity of PNA recognition. As with DNAhybridization, however, selective recognition can be achieved bybalancing oligomer length and incubation temperature. Moreover,selective hybridization of PNAs is encouraged by PNA-DNA hybridizationbeing less tolerant of base mismatches than DNA-DNA hybridization. Forexample, a single mismatch within a 16 bp PNA-DNA duplex can reduce theT_(m) by up to 15° C. (Egholm et al., 1993). This high level ofdiscrimination has allowed the development of several PNA-basedstrategies for the analysis of point mutations (Wang et al., 1996;Carlsson et al., 1996; Thiede et al., 1996; Webb and Hurskainen, 1996;Perry-O'Keefe et al., 1996).

High-affinity binding provides clear advantages for molecularrecognition and the development of new applications for PNAs. Forexample, 11-13 nucleotide PNAs inhibit the activity of telomerase, aribonucleo-protein that extends telomere ends using an essential RNAtemplate, while the analogous DNA oligomers do not (Norton et al.,1996).

Neutral PNAs are more hydrophobic than analogous DNA oligomers, and thiscan lead to difficulty solubilizing them at neutral pH, especially ifthe PNAs have a high purine content or if they have the potential toform secondary structures. Their solubility can be enhanced by attachingone or more positive charges to the PNA termini (Nielsen et al., 1991).

Findings by Allfrey and colleagues suggest that strand invasion willoccur spontaneously at sequences within chromosomal DNA (Boffa et al.,1995; Boffa et al., 1996). These studies targeted PNAs to tripletrepeats of the nucleotides CAG and used this recognition to purifytranscriptionally active DNA (Boffa et al., 1995) and to inhibittranscription (Boffa et al., 1996). This result suggests that if PNAscan be delivered within cells then they will have the potential to begeneral sequence-specific regulators of gene expression. Studies andreviews concerning the use of PNAs as antisense and anti-gene agentsinclude Nielsen et al. (1993b), Hanvey et al. (1992), and Good andNielsen (1997). Koppelhus et al. (1997) have used PNAs to inhibit HIV-1inverse transcription, showing that PNAs may be used for antiviraltherapies.

Methods of characterizing the antisense binding properties of PNAs arediscussed in Rose (1993) and Jensen et al. (1997). Rose uses capillarygel electrophoresis to determine binding of PNAs to their complementaryoligonucleotide, measuring the relative binding kinetics andstoichiometry. Similar types of measurements were made by Jensen et al.using BIAcore™ technology.

Other applications of PNAs include use in DNA strand invasion (Nielsenet al., 1991), antisense inhibition (Hanvey et al., 1992), mutationalanalysis (Orum et al., 1993), enhancers of transcription (Mollegaard etal., 1994), nucleic acid purification (Orum et al., 1995), isolation oftranscriptionally active genes (Boffa et al., 1995), blocking oftranscription factor binding (Vickers et al., 1995), genome cleavage(Veselkov et al., 1996), biosensors (Wang et al., 1996), in situhybridization (Thisted et al., 1996), and in a alternative to Southernblotting (Perry-O'Keefe, 1996).

POLYPEPTIDE COMPOSITIONS

The present invention, in other aspects, provides polypeptidecompositions. Generally, a polypeptide of the invention will be anisolated polypeptide (or an epitope, variant, or active fragmentthereof) derived from a mammalian species. Preferably, the polypeptideis encoded by a polynucleotide sequence disclosed herein or a sequencewhich hybridizes under moderately stringent conditions to apolynucleotide sequence disclosed herein. Alternatively, the polypeptidemay be defined as a polypeptide which comprises a contiguous amino acidsequence from an amino acid sequence disclosed herein, or whichpolypeptide comprises an entire amino acid sequence disclosed herein.

In the present invention, a polypeptide composition is also understoodto comprise one or more polypeptides that are immunologically reactivewith antibodies generated against a polypeptide of the invention,particularly a polypeptide having the amino acid sequence disclosed inSEQ ID NO: 324-340, 783, 786, 787, 789, 791, 793, 795, 797-799, 805,806, 809, 827, 1667, 1670-1675, 1677-1679, 1806-1822, 1825 or to activefragments, or to variants or biological functional equivalents thereof.

Likewise, a polypeptide composition of the present invention isunderstood to comprise one or more polypeptides that are capable ofeliciting antibodies that are immunologically reactive with one or morepolypeptides encoded by one or more contiguous nucleic acid sequencescontained in SEQ ID NO: 1-323, 341-782, 784-785, 788, 790, 792, 794,796, 800-804, 807, 808, 810-826, 878-1664, 1668, 1669, 1676, 1680-1805,1824, or to active fragments, or to variants thereof, or to one or morenucleic acid sequences which hybridize to one or more of these sequencesunder conditions of moderate to high stringency. Particularlyillustrative polypeptides include the amino acid sequences disclosed inSEQ ID NO: 324-340, 783, 786, 787, 789, 791, 793, 795, 797-799, 805,806, 809, 827, 1667, 1670-1675, 1677-1679, 1806-1822 and 1825.

As used herein, an active fragment of a polypeptide includes a whole ora portion of a polypeptide which is modified by conventional techniques,e.g., mutagenesis, or by addition, deletion, or substitution, but whichactive fragment exhibits substantially the same structure function,antigenicity, etc., as a polypeptide as described herein.

In certain illustrative embodiments, the polypeptides of the inventionwill comprise at least an immunogenic portion of a lung tumor protein ora variant thereof, as described herein. As noted above, a “lung tumorprotein” is a protein that is expressed by lung tumor cells. Proteinsthat are lung tumor proteins also react detectably within an immunoassay(such as an ELISA) with antisera from a patient with lung cancer.Polypeptides as described herein may be of any length. Additionalsequences derived from the native protein and/or heterologous sequencesmay be present, and such sequences may (but need not) possess furtherimmunogenic or antigenic properties.

An “immunogenic portion,” as used herein is a portion of a protein thatis recognized (i.e., specifically bound) by a B-cell and/or T-cellsurface antigen receptor. Such immunogenic portions generally compriseat least 5 amino acid residues, more preferably at least 10, and stillmore preferably at least 20 amino acid residues of a lung tumor proteinor a variant thereof. Certain preferred immunogenic portions includepeptides in which an N-terminal leader sequence and/or transmembranedomain have been deleted. Other preferred immunogenic portions maycontain a small N- and/or C-terminal deletion (e.g., 1-30 amino acids,preferably 5-15 amino acids), relative to the mature protein.

Immunogenic portions may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Suchtechniques include screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well known techniques. An immunogenic portion of anative lung tumor protein is a portion that reacts with such antiseraand/or T-cells at a level that is not substantially less than thereactivity of the full length polypeptide (e.g., in an ELISA and/orT-cell reactivity assay). Such immunogenic portions may react withinsuch assays at a level that is similar to or greater than the reactivityof the full length polypeptide. Such screens may generally be performedusing methods well known to those of ordinary skill in the art, such asthose described in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. For example, a polypeptide may beimmobilized on a solid support and contacted with patient sera to allowbinding of antibodies within the sera to the immobilized polypeptide.Unbound sera may then be removed and bound antibodies detected using,for example, ¹²⁵I-labeled Protein A.

As noted above, a composition may comprise a variant of a native lungtumor protein. A polypeptide “variant,” as used herein, is a polypeptidethat differs from a native lung tumor protein in one or moresubstitutions, deletions, additions and/or insertions, such that theimmunogenicity of the polypeptide is not substantially diminished. Inother words, the ability of a variant to react with antigen-specificantisera may be enhanced or unchanged, relative to the native protein,or may be diminished by less than 50%, and preferably less than 20%,relative to the native protein. Such variants may generally beidentified by modifying one of the above polypeptide sequences andevaluating the reactivity of the modified polypeptide withantigen-specific antibodies or antisera as described herein. Preferredvariants include those in which one or more portions, such as anN-terminal leader sequence or transmembrane domain, have been removed.Other preferred variants include variants in which a small portion(e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removedfrom the N- and/or C-terminal of the mature protein.

Polypeptide variants encompassed by the present invention include thoseexhibiting at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% or more identity (determined as describedabove) to the polypeptides disclosed herein.

Preferably, a variant contains conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. Amino acid substitutions may generally be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. In a preferredembodiment, variant polypeptides differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer.Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theimmunogenicity, secondary structure and hydropathic nature of thepolypeptide.

As noted above, polypeptides may comprise a signal (or leader) sequenceat the N-terninal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

Polypeptides may be prepared using any of a variety of well knowntechniques. Recombinant polypeptides encoded by DNA sequences asdescribed above may be readily prepared from the DNA sequences using anyof a variety of expression vectors known to those of ordinary skill inthe art. Expression may be achieved in any appropriate host cell thathas been transformed or transfected with an expression vector containinga DNA molecule that encodes a recombinant polypeptide. Suitable hostcells include prokaryotes, yeast, and higher eukaryotic cells, such asmammalian cells and plant cells. Preferably, the host cells employed areE. coli, yeast or a mammalian cell line such as COS or CHO. Supernatantsfrom suitable host/vector systems which secrete recombinant protein orpolypeptide into culture media may be first concentrated using acommercially available filter. Following concentration, the concentratemay be applied to a suitable purification matrix such as an affinitymatrix or an ion exchange resin. Finally, one or more reverse phase HPLCsteps can be employed to further purify a recombinant polypeptide.

Portions and other variants having less than about 100 amino acids, andgenerally less than about 50 amino acids, may also be generated bysynthetic means, using techniques well known to those of ordinary skillin the art. For example, such polypeptides may be synthesized using anyof the commercially available solid-phase techniques, such as theMerrifield solid-phase synthesis method, where amino acids aresequentially added to a growing amino acid chain. See Merrifield, J. Am.Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City, Calif.), and may beoperated according to the manufacturer's instructions.

Within certain specific embodiments, a polypeptide may be a fusionprotein that comprises multiple polypeptides as described herein, orthat comprises at least one polypeptide as described herein and anunrelated sequence, such as a known tumor protein. A fusion partner may,for example, assist in providing T helper epitopes (an immunologicalfusion partner), preferably T helper epitopes recognized by humans, ormay assist in expressing the protein (an expression enhancer) at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the protein or to enable the protein to be targeted todesired intracellular compartments. Still further fusion partnersinclude affinity tags, which facilitate purification of the protein.

Fusion proteins may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion protein isexpressed as a recombinant protein, allowing the production of increasedlevels, relative to a non-fused protein, in an expression system.Briefly, DNA sequences encoding the polypeptide components may beassembled separately, and ligated into an appropriate expression vector.The 3′ end of the DNA sequence encoding one polypeptide component isligated, with or without a peptide linker, to the 5′ end of a DNAsequence encoding the second polypeptide component so that the readingframes of the sequences are in phase. This permits translation into asingle fusion protein that retains the biological activity of bothcomponent polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may generally be from 1 to about 50 amino acids inlength. Linker sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

Fusion proteins are also provided. Such proteins comprise a polypeptideas described herein together with an unrelated immunogenic protein.Preferably the immunogenic protein is capable of eliciting a recallresponse. Examples of such proteins include tetanus, tuberculosis andhepatitis proteins (see, for example, Stoute et al. New Engl. J. Med.,336:86-91, 1997).

Within preferred embodiments, an immunological fusion partner is derivedfrom protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292, 1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionprotein. A repeat portion is found in the C-terminal region starting atresidue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

In general, polypeptides (including fusion proteins) and polynucleotidesas described herein are isolated. An “isolated” polypeptide orpolynucleotide is one that is removed from its original environment. Forexample, a naturally-occurring protein is isolated if it is separatedfrom some or all of the coexisting materials in the natural system.Preferably, such polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not a part of the naturalenvironment.

BINDING AGENTS

The present invention further provides agents, such as antibodies andantigen-binding fragments thereof, that specifically bind to a lungtumor protein. As used herein, an antibody, or antigen-binding fragmentthereof, is said to “specifically bind” to a lung tumor protein if itreacts at a detectable level (within, for example, an ELISA) with a lungtumor protein, and does not react detectably with unrelated proteinsunder similar conditions. As used herein, “binding” refers to anoncovalent association between two separate molecules such that acomplex is formed. The ability to bind may be evaluated by, for example,determining a binding constant for the formation of the complex. Thebinding constant is the value obtained when the concentration of thecomplex is divided by the product of the component concentrations. Ingeneral, two compounds are said to “bind,” in the context of the presentinvention, when the binding constant for complex formation exceeds about10³ L/mol. The binding constant may be determined using methods wellknown in the art.

Binding agents may be further capable of differentiating betweenpatients with and without a cancer, such as lung cancer, using therepresentative assays provided herein. In other words, antibodies orother binding agents that bind to a lung tumor protein will generate asignal indicating the presence of a cancer in at least about 20% ofpatients with the disease, and will generate a negative signalindicating the absence of the disease in at least about 90% ofindividuals without the cancer. To determine whether a binding agentsatisfies this requirement, biological samples (e.g., blood, sera,sputum, urine and/or tumor biopsies) from patients with and without acancer (as determined using standard clinical tests) may be assayed asdescribed herein for the presence of polypeptides that bind to thebinding agent. It will be apparent that a statistically significantnumber of samples with and without the disease should be assayed. Eachbinding agent should satisfy the above criteria; however, those ofordinary skill in the art will recognize that binding agents may be usedin combination to improve sensitivity.

Any agent that satisfies the above requirements may be a binding agent.For example, a binding agent may be a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, antibodies can be produced by cell culturetechniques, including the generation of monoclonal antibodies asdescribed herein, or via transfection of antibody genes into suitablebacterial or mammalian cell hosts, in order to allow for the productionof recombinant antibodies. In one technique, an immunogen comprising thepolypeptide is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep or goats). In this step, thepolypeptides of this invention may serve as the immunogen withoutmodification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for an antigenic polypeptide of interestmay be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

Within certain embodiments, the use of antigen-binding fragments ofantibodies may be preferred. Such fragments include Fab fragments, whichmay be prepared using standard techniques. Briefly, immunoglobulins maybe purified from rabbit serum by affinity chromatography on Protein Abead columns (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988) and digested by papain to yield Fab andFc fragments. The Fab and Fc fragments may be separated by affinitychromatography on protein A bead columns.

Monoclonal antibodies of the present invention may be coupled to one ormore therapeutic agents. Suitable agents in this regard includeradionuclides, differentiation inducers, drugs, toxins, and derivativesthereof. Preferred radionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re,¹⁸⁸Re, ²¹¹At, and ²¹²Bi. Preferred drugs include methotrexate, andpyrimidine and purine analogs. Preferred differentiation inducersinclude phorbol esters and butyric acid. Preferred toxins include ricin,abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin,Shigella toxin, and pokeweed antiviral protein.

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used. Alternatively, a carrier can be used.

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group. Suitable carriers includeproteins such as albumins (e.g., U.S. Pat. No. 4,507,234, to Kato etal.), peptides and polysaccharides such as aminodextran (e.g., U.S. Pat.No. 4,699,784, to Shih et al.). A carrier may also bear an agent bynoncovalent bonding or by encapsulation, such as within a liposomevesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriersspecific for radionuclide agents include radiohalogenated smallmolecules and chelating compounds. For example, U.S. Pat. No. 4,735,792discloses representative radiohalogenated small molecules and theirsynthesis. A radionuclide chelate may be formed from chelating compoundsthat include those containing nitrogen and sulfur atoms as the donoratoms for binding the metal, or metal oxide, radionuclide. For example,U.S. Pat. No. 4,673,562, to Davison et al. discloses representativechelating compounds and their synthesis.

A variety of routes of administration for the antibodies andimmunoconjugates may be used. Typically, administration will beintravenous, intramuscular, subcutaneous or in the bed of a resectedtumor. It will be evident that the precise dose of theantibody/immunoconjugate will vary depending upon the antibody used, theantigen density on the tumor, and the rate of clearance of the antibody.

T CELLS

Immunotherapeutic compositions may also, or alternatively, comprise Tcells specific for a lung tumor protein. Such cells may generally beprepared in vitro or ex vivo, using standard procedures. For example, Tcells may be isolated from bone marrow, peripheral blood, or a fractionof bone marrow or peripheral blood of a patient, using a commerciallyavailable cell separation system, such as the Isolex™ System, availablefrom Nexell Therapeutics, Inc. (Irvine, Calif.; see also U.S. Pat. No.5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO92/07243). Alternatively, T cells may be derived from related orunrelated humans, non-human mammals, cell lines or cultures.

T cells may be stimulated with a lung tumor polypeptide, polynucleotideencoding a lung tumor polypeptide and/or an antigen presenting cell(APC) that expresses such a polypeptide. Such stimulation is performedunder conditions and for a time sufficient to permit the generation of Tcells that are specific for the polypeptide. Preferably, a lung tumorpolypeptide or polynucleotide is present within a delivery vehicle, suchas a microsphere, to facilitate the generation of specific T cells.

T cells are considered to be specific for a lung tumor polypeptide ifthe T cells specifically proliferate, secrete cytokines or kill targetcells coated with the polypeptide or expressing a gene encoding thepolypeptide. T cell specificity may be evaluated using any of a varietyof standard techniques. For example, within a chromium release assay orproliferation assay, a stimulation index of more than two fold increasein lysis and/or proliferation, compared to negative controls, indicatesT cell specificity. Such assays may be performed, for example, asdescribed in Chen et al., Cancer Res. 54:1065-1070, 1994. Alternatively,detection of the proliferation of T cells may be accomplished by avariety of known techniques. For example, T cell proliferation can bedetected by measuring an increased rate of DNA synthesis (e.g., bypulse-labeling cultures of T cells with tritiated thymidine andmeasuring the amount of tritiated thymidine incorporated into DNA).Contact with a lung tumor polypeptide (100 ng/ml-100 μg/ml, preferably200 ng/ml-25 μg/ml) for 3-7 days should result in at least a two foldincrease in proliferation of the T cells. Contact as described above for2-3 hours should result in activation of the T cells, as measured usingstandard cytokine assays in which a two fold increase in the level ofcytokine release (e.g., TNF or IFN-γ) is indicative of T cell activation(see Coligan et al., Current Protocols in Immunology, vol. 1, WileyInterscience (Greene 1998)). T cells that have been activated inresponse to a lung tumor polypeptide, polynucleotide orpolypeptide-expressing APC may be CD4⁺ and/or CD8⁺. Lung tumorprotein-specific T cells may be expanded using standard techniques.Within preferred embodiments, the T cells are derived from a patient, arelated donor or an unrelated donor, and are administered to the patientfollowing stimulation and expansion.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate inresponse to a lung tumor polypeptide, polynucleotide or APC can beexpanded in number either in vitro or in vivo. Proliferation of such Tcells in vitro may be accomplished in a variety of ways. For example,the T cells can be re-exposed to a lung tumor polypeptide, or a shortpeptide corresponding to an immunogenic portion of such a polypeptide,with or without the addition of T cell growth factors, such asinterleukin-2, and/or stimulator cells that synthesize a lung tumorpolypeptide. Alternatively, one or more T cells that proliferate in thepresence of a lung tumor protein can be expanded in number by cloning.Methods for cloning cells are well known in the art, and includelimiting dilution.

PHARMACEUTICAL COMPOSITIONS

In additional embodiments, the present invention concerns formulation ofone or more of the polynucleotide, polypeptide, T-cell and/or antibodycompositions disclosed herein in pharmaceutically-acceptable solutionsfor administration to a cell or an animal, either alone, or incombination with one or more other modalities of therapy.

It will also be understood that, if desired, the nucleic acid segment,RNA, DNA or PNA compositions that express a polypeptide as disclosedherein may be administered in combination with other agents as well,such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The compositions may thus be deliveredalong with various other agents as required in the particular instance.Such compositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

1. ORAL DELIVERY

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al.,1997; Hwang et al., 1998; U.S. Pat. No. 5,641,515; U.S. Pat. No.5,580,579 and U.S. Pat. No. 5,792,451, each specifically incorporatedherein by reference in its entirety). The tablets, troches, pills,capsules and the like may also contain the following: a binder, as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.A syrup of elixir may contain the active compound sucrose as asweetening agent methyl and propylparabens as preservatives, a dye andflavoring, such as cherry or orange flavor. Of course, any material usedin preparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

2. INJECTABLE DELIVERY

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally as describedin U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No.5,399,363 (each specifically incorporated herein by reference in itsentirety). Solutions of the active compounds as free base orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug-release capsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

3. NASAL DELIVERY

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, nucleic acids, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212(each specifically incorporated herein by reference in its entirety).Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S.Pat. No. 5,725,871, specifically incorporated herein by reference in itsentirety) are also well-known in the pharmaceutical arts. Likewise,transmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045 (specificallyincorporated herein by reference in its entirety).

4. LIPOSOME-, NANOCAPSULE-, AND MICROPARTICLE-MEDIATED DELIVERY

In certain embodiments, the inventors contemplate the use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, or ananoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically-acceptable formulations of the nucleic acids orconstructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art (see for example, Couvreuret al., 1977; Couvreur, 1988; Lasic, 1998; which describes the use ofliposomes and nanocapsules in the targeted antibiotic therapy forintracellular bacterial infections and diseases). Recently, liposomeswere developed with improved serum stability and circulation half-times(Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987; U.S. Pat. No.5,741,516, specifically incorporated herein by reference in itsentirety). Further, various methods of liposome and liposome likepreparations as potential drug carriers have been reviewed (Takakura,1998; Chandran et al., 1997; Margalit, 1995; U.S. Pat. No. 5,567,434;U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No.5,738,868 and U.S. Pat. No. 5,795,587, each specifically incorporatedherein by reference in its entirety).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., 1990; Muller et al., 1990). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes, drugs(Heath and Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989;Fresta and Puglisi, 1996), radiotherapeutic agents (Pikul et al., 1987),enzymes (Imaizumi et al., 1990a; Imaizumi et al., 1990b), viruses(Faller and Baltimore, 1984), transcription factors and allostericeffectors (Nicolau and Gersonde, 1979) into a variety of cultured celllines and animals. In addition, several successful clinical trailsexamining the effectiveness of liposome-mediated drug delivery have beencompleted (Lopez-Berestein et al., 1985a; 1985b; Coune, 1988; Sculier etal., 1988). Furthermore, several studies suggest that the use ofliposomes is not associated with autoimmune responses, toxicity orgonadal localization after systemic delivery (Mori and Fukatsu, 1992).

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Liposomes bear resemblance to cellular membranes and are contemplatedfor use in connection with the present invention as carriers for thepeptide compositions. They are widely suitable as both water- andlipid-soluble substances can be entrapped, i.e. in the aqueous spacesand within the bilayer itself, respectively. It is possible that thedrug-bearing liposomes may even be employed for site-specific deliveryof active agents by selectively modifying the liposomal formulation.

In addition to the teachings of Couvreur et al. (1977; 1988), thefollowing information may be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

In addition to temperature, exposure to proteins can alter thepermeability of liposomes. Certain soluble proteins, such as cytochromec, bind, deform and penetrate the bilayer, thereby causing changes inpermeability. Cholesterol inhibits this penetration of proteins,apparently by packing the phospholipids more tightly. It is contemplatedthat the most useful liposome formations for antibiotic and inhibitordelivery will contain cholesterol.

The ability to trap solutes varies between different types of liposomes.For example, MLVs are moderately efficient at trapping solutes, but SUVsare extremely inefficient. SUVs offer the advantage of homogeneity andreproducibility in size distribution, however, and a compromise betweensize and trapping efficiency is offered by large unilamellar vesicles(LUVs). These are prepared by ether evaporation and are three to fourtimes more efficient at solute entrapment than MLVs.

In addition to liposome characteristics, an important determinant inentrapping compounds is the physicochemical properties of the compounditself. Polar compounds are trapped in the aqueous spaces and nonpolarcompounds bind to the lipid bilayer of the vesicle. Polar compounds arereleased through permeation or when the bilayer is broken, but nonpolarcompounds remain affiliated with the bilayer unless it is disrupted bytemperature or exposure to lipoproteins. Both types show maximum effluxrates at the phase transition temperature.

Liposomes interact with cells via four different mechanisms: endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. It often is difficult to determine which mechanism isoperative and more than one may operate at the same time.

The fate and disposition of intravenously injected liposomes depend ontheir physical properties, such as size, fluidity, and surface charge.They may persist in tissues for h or days, depending on theircomposition, and half lives in the blood range from min to several h.Larger liposomes, such as MLVs and LUVs, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominate site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehavior limits the potential targeting of liposomes to only thoseorgans and tissues accessible to their large size. These include theblood, liver, spleen, bone marrow, and lymphoid organs.

Targeting is generally not a limitation in terms of the presentinvention. However, should specific targeting be desired, methods areavailable for this to be accomplished. Antibodies may be used to bind tothe liposome surface and to direct the antibody and its drug contents tospecific antigenic receptors located on a particular cell-type surface.Carbohydrate determinants (glycoprotein or glycolipid cell-surfacecomponents that play a role in cell-cell recognition, interaction andadhesion) may also be used as recognition sites as they have potentialin directing liposomes to particular cell types. Mostly, it iscontemplated that intravenous injection of liposomal preparations wouldbe used, but other routes of administration are also conceivable.

Alternatively, the invention provides for pharmaceutically-acceptablenanocapsule formulations of the compositions of the present invention.Nanocapsules can generally entrap compounds in a stable and reproducibleway (Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998;Douglas et al., 1987). To avoid side effects due to intracellularpolymeric overloading, such ultrafine particles (sized around 0.1 μm)should be designed using polymers able to be degraded in vivo.Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet theserequirements are contemplated for use in the present invention. Suchparticles may be are easily made, as described (Couvreur et al., 1980;1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry etal., 1995 and U.S. Pat. No. 5,145,684, specifically incorporated hereinby reference in its entirety).

IMMUNOGENIC COMPOSITIONS

In certain preferred embodiments of the present invention, immunogeniccompositions, or vaccines, are provided. The immunogenic compositionswill generally comprise one or more pharmaceutical compositions, such asthose discussed above, in combination with an immunostimulant. Animmunostimulant may be any substance that enhances or potentiates animmune response (antibody and/or cell-mediated) to an exogenous antigen.Examples of immunostimulants include adjuvants, biodegradablemicrospheres (e.g., polylactic galactide) and liposomes (into which thecompound is incorporated; see e.g., Fullerton, U.S. Pat. No. 4,235,877).Vaccine preparation is generally described in, for example, M. F. Powelland M. J. Newman, eds., “Vaccine Design (the subunit and adjuvantapproach),” Plenum Press (NY, 1995). Pharmaceutical compositions andimmunogenic compositions, or vaccines, within the scope of the presentinvention may also contain other compounds, which may be biologicallyactive or inactive. For example, one or more immunogenic portions ofother tumor antigens may be present, either incorporated into a fusionpolypeptide or as a separate compound, within the composition.

Illustrative immunogenic compositions may contain DNA encoding one ormore of the polypeptides as described above, such that the polypeptideis generated in situ. As noted above, the DNA may be present within anyof a variety of delivery systems known to those of ordinary skill in theart, including nucleic acid expression systems, bacteria and viralexpression systems. Numerous gene delivery techniques are well known inthe art, such as those described by Rolland, Crit. Rev. Therap. DrugCarrier Systems 15:143-198, 1998, and references cited therein.Appropriate nucleic acid expression systems contain the necessary DNAsequences for expression in the patient (such as a suitable promoter andterminating signal). Bacterial delivery systems involve theadministration of a bacterium (such as Bacillus-Calmette-Guerrin) thatexpresses an immunogenic portion of the polypeptide on its cell surfaceor secretes such an epitope. In a preferred embodiment, the DNA may beintroduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus. Suitablesystems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl.Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci.569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos.4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434,1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994;Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993;Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir.Res. 73:1202-1207, 1993. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.The DNA may also be “naked,” as described, for example, in Ulmer et al.,Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells. It will be apparent that an immunogenic composition maycomprise both a polynucleotide and a polypeptide component. Suchimmunogenic compositions may provide for an enhanced immune response.

It will be apparent that an immunogenic composition may containpharmaceutically acceptable salts of the polynucleotides andpolypeptides provided herein. Such salts may be prepared frompharmaceutically acceptable non-toxic bases, including organic bases(e.g., salts of primary, secondary and tertiary amines and basic aminoacids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium,calcium and magnesium salts).

While any suitable carrier known to those of ordinary skill in the artmay be employed in the immunogenic compositions of this invention, thetype of carrier will vary depending on the mode of administration.Compositions of the present invention may be formulated for anyappropriate manner of administration, including for example, topical,oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763;5,814,344 and 5,942,252. One may also employ a carrier comprising theparticulate-protein complexes described in U.S. Pat. No. 5,928,647,which are capable of inducing a class I-restricted cytotoxic Tlymphocyte responses in a host.

Such compositions may also comprise buffers (e.g., neutral bufferedsaline or phosphate buffered saline), carbohydrates (e.g., glucose,mannose, sucrose or dextrans), mannitol, proteins, polypeptides or aminoacids such as glycine, antioxidants, bacteriostats, chelating agentssuch as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide),solutes that render the formulation isotonic, hypotonic or weaklyhypertonic with the blood of a recipient, suspending agents, thickeningagents and/or preservatives. Alternatively, compositions of the presentinvention may be formulated as a lyophilizate. Compounds may also beencapsulated within liposomes using well known technology.

Any of a variety of immunostimulants may be employed in the immunogeniccompositions of this invention. For example, an adjuvant may beincluded. Most adjuvants contain a substance designed to protect theantigen from rapid catabolism, such as aluminum hydroxide or mineraloil, and a stimulator of immune responses, such as lipid A, Bortadellapertussis or Mycobacterium tuberculosis derived proteins. Suitableadjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2(SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminumhydroxide gel (alum) or aluminum phosphate; salts of calcium, iron orzinc; an insoluble suspension of acylated tyrosine; acylated sugars;cationically or anionically derivatized polysaccharides;polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A andquil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may alsobe used as adjuvants.

Within the immunogenic compositions provided herein, the adjuvantcomposition is preferably designed to induce an immune responsepredominantly of the Th1 type. High levels of Th1-type cytokines (e.g.,IFN-γ, TNFα, IL-2 and IL-12) tend to favor the induction of cellmediated immune responses to an administered antigen. In contrast, highlevels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend tofavor the induction of humoral immune responses. Following applicationof an immunogenic composition as provided herein, a patient will supportan immune response that includes Th1- and Th2-type responses. Within apreferred embodiment, in which a response is predominantly Th1-type, thelevel of Th1-type cytokines will increase to a greater extent than thelevel of Th2-type cytokines. The levels of these cytokines may bereadily assessed using standard assays. For a review of the families ofcytokines, see Mosmann and Coffinan, Ann. Rev. Immunol. 7:145-173, 1989.

Preferred adjuvants for use in eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), togetherwith an aluminum salt. MPL adjuvants are available from CorixaCorporation (Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc.,Framingham, Mass.), which may be used alone or in combination with otheradjuvants. For example, an enhanced system involves the combination of amonophosphoryl lipid A and saponin derivative, such as the combinationof QS21 and 3D-MPL as described in WO 94/00153, or a less reactogeniccomposition where the QS21 is quenched with cholesterol, as described inWO 96/33739. Other preferred formulations comprise an oil-in-wateremulsion and tocopherol. A particularly potent adjuvant formulationinvolving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion isdescribed in WO 95/17210.

Other preferred adjuvants include Montanide ISA 720 (Seppic, France),SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), theSBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available fromSmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton,Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkylglucosaminide 4-phosphates (AGPs), such as those described in pendingU.S. patent application Ser. Nos. 08/853,826 and 09/074,720, thedisclosures of which are incorporated herein by reference in theirentireties.

Any immunogenic composition provided herein may be prepared using wellknown methods that result in a combination of antigen, immune responseenhancer and a suitable carrier or excipient. The compositions describedherein may be administered as part of a sustained release formulation(i.e., a formulation such as a capsule, sponge or gel (composed ofpolysaccharides, for example) that effects a slow release of compoundfollowing administration). Such formulations may generally be preparedusing well known technology (see, e.g., Coombes et al., Vaccine14:1429-1438, 1996) and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.

Carriers for use within such formulations are biocompatible, and mayalso be biodegradable; preferably the formulation provides a relativelyconstant level of active component release. Such carriers includemicroparticles of poly(lactide-co-glycolide), polyacrylate, latex,starch, cellulose, dextran and the like. Other delayed-release carriersinclude supramolecular biovectors, which comprise a non-liquidhydrophilic core (e.g., a cross-linked polysaccharide oroligosaccharide) and, optionally, an external layer comprising anamphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO96/06638). The amount of active compound contained within a sustainedrelease formulation depends upon the site of implantation, the rate andexpected duration of release and the nature of the condition to betreated or prevented.

Any of a variety of delivery vehicles may be employed withinpharmaceutical compositions and immunogenic compositions to facilitateproduction of an antigen-specific immune response that targets tumorcells. Delivery vehicles include antigen presenting cells (APCs), suchas dendritic cells, macrophages, B cells, monocytes and other cells thatmay be engineered to be efficient APCs. Such cells may, but need not, begenetically modified to increase the capacity for presenting theantigen, to improve activation and/or maintenance of the T cellresponse, to have anti-tumor effects per se and/or to be immunologicallycompatible with the receiver (i.e., matched HLA haplotype). APCs maygenerally be isolated from any of a variety of biological fluids andorgans, including tumor and peritumoral tissues, and may be autologous,allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaive T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine, or immunogeniccomposition (see Zitvogel et al., Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide encoding a lungtumor protein (or portion or other variant thereof) such that the lungtumor polypeptide, or an immunogenic portion thereof, is expressed onthe cell surface. Such transfection may take place ex vivo, and acomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the lung tumor polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

Immunogenic compositions and pharmaceutical compositions may bepresented in unit-dose or multi-dose containers, such as sealed ampoulesor vials. Such containers are preferably hermetically sealed to preservesterility of the formulation until use. In general, formulations may bestored as suspensions, solutions or emulsions in oily or aqueousvehicles. Alternatively, an immunogenic or pharmaceutical compositionmay be stored in a freeze-dried condition requiring only the addition ofa sterile liquid carrier immediately prior to use.

CANCER THERAPY

In further aspects of the present invention, the compositions describedherein may be used for immunotherapy of cancer, such as lung cancer.Within such methods, compositions are typically administered to apatient. As used herein, a “patient” refers to any warm-blooded animal,preferably a human. A patient may or may not be afflicted with cancer.Accordingly, the above pharmaceutical compositions and immunogeniccompositions may be used to prevent the development of a cancer or totreat a patient afflicted with a cancer. A cancer may be diagnosed usingcriteria generally accepted in the art, including the presence of amalignant tumor. Pharmaceutical compositions and immunogeniccompositions may be administered either prior to or following surgicalremoval of primary tumors and/or treatment such as administration ofradiotherapy or conventional chemotherapeutic drugs. Administration maybe by any suitable method, including administration by intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal, intradennal,anal, vaginal, topical and oral routes.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against tumors with the administration ofimmune response-modifying agents (such as polypeptides andpolynucleotides as provided herein).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedtumor-immune reactivity (such as effector cells or antibodies) that candirectly or indirectly mediate antitumor effects and does notnecessarily depend on an intact host immune system. Examples of effectorcells include T cells as discussed above, T lymphocytes (such as CD8⁺cytotoxic T lymphocytes and CD4⁺ T-helper tumor-infiltratinglymphocytes), killer cells (such as Natural Killer cells andlymphokine-activated killer cells), B cells and antigen-presenting cells(such as dendritic cells and macrophages) expressing a polypeptideprovided herein. T cell receptors and antibody receptors specific forthe polypeptides recited herein may be cloned, expressed and transferredinto other vectors or effector cells for adoptive immunotherapy. Thepolypeptides provided herein may also be used to generate antibodies oranti-idiotypic antibodies (as described above and in U.S. Pat. No.4,918,164) for passive immunotherapy.

Effector cells may generally be obtained in sufficient quantities foradoptive immunotherapy by growth in vitro, as described herein. Cultureconditions for expanding single antigen-specific effector cells toseveral billion in number with retention of antigen recognition in vivoare well known in the art. Such in vitro culture conditions typicallyuse intermittent stimulation with antigen, often in the presence ofcytokines (such as IL-2) and non-dividing feeder cells. As noted above,immunoreactive polypeptides as provided herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate asufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage, monocyte,fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., Immunological Reviews 157:177, 1997).

Alternatively, a vector expressing a polypeptide recited herein may beintroduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary, intraperitoneal or intratumor administration.

Routes and frequency of administration of the therapeutic compositionsdescribed herein, as well as dosage, will vary from individual toindividual, and may be readily established using standard techniques. Ingeneral, the pharmaceutical compositions and immunogenic compositionsmay be administered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Preferably, between 1 and 10 doses may be administered over a 52week period. Preferably, 6 doses are administered, at intervals of 1month, and booster vaccinations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients. Asuitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an anti-tumor immune response,and is at least 10-50% above the basal (i.e., untreated) level. Suchresponse can be monitored by measuring the anti-tumor antibodies in apatient or by vaccine-dependent generation of cytolytic effector cellscapable of killing the patient's tumor cells in vitro. Such vaccines, orimmunogenic compositions, should also be capable of causing an immuneresponse that leads to an improved clinical outcome (e.g., more frequentremissions, complete or partial or longer disease-free survival) invaccinated patients as compared to non-vaccinated patients. In general,for compositions comprising one or more polypeptides, the amount of eachpolypeptide present in a dose ranges from about 25 μg to 5 mg per kg ofhost. Suitable dose sizes will vary with the size of the patient, butwill typically range from about 0.1 mL to about 5 mL.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., more frequent remissions, completeor partial, or longer disease-free survival) in treated patients ascompared to non-treated patients. Increases in preexisting immuneresponses to a lung tumor protein generally correlate with an improvedclinical outcome. Such immune responses may generally be evaluated usingstandard proliferation, cytotoxicity or cytokine assays, which may beperformed using samples obtained from a patient before and aftertreatment.

CANCER DETECTION AND DIAGNOSIS

In general, a cancer may be detected in a patient based on the presenceof one or more lung tumor proteins and/or polynucleotides encoding suchproteins in a biological sample (for example, blood, sera, sputum urineand/or tumor biopsies) obtained from the patient. In other words, suchproteins may be used as markers to indicate the presence or absence of acancer such as lung cancer. In addition, such proteins may be useful forthe detection of other cancers. The binding agents provided hereingenerally permit detection of the level of antigen that binds to theagent in the biological sample. Polynucleotide primers and probes may beused to detect the level of mRNA encoding a tumor protein, which is alsoindicative of the presence or absence of a cancer. In general, a lungtumor sequence should be present at a level that is at least three foldhigher in tumor tissue than in normal tissue There are a variety ofassay formats known to those of ordinary skill in the art for using abinding agent to detect polypeptide markers in a sample. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, the presence or absence of a cancer in apatient may be determined by (a) contacting a biological sample obtainedfrom a patient with a binding agent; (b) detecting in the sample a levelof polypeptide that binds to the binding agent; and (c) comparing thelevel of polypeptide with a predetermined cut-off value.

In a preferred embodiment, the assay involves the use of binding agentimmobilized on a solid support to bind to and remove the polypeptidefrom the remainder of the sample. The bound polypeptide may then bedetected using a detection reagent that contains a reporter group andspecifically binds to the binding agent/polypeptide complex. Suchdetection reagents may comprise, for example, a binding agent thatspecifically binds to the polypeptide or an antibody or other agent thatspecifically binds to the binding agent, such as an anti-immunoglobulin,protein G, protein A or a lectin. Alternatively, a competitive assay maybe utilized, in which a polypeptide is labeled with a reporter group andallowed to bind to the immobilized binding agent after incubation of thebinding agent with the sample. The extent to which components of thesample inhibit the binding of the labeled polypeptide to the bindingagent is indicative of the reactivity of the sample with the immobilizedbinding agent. Suitable polypeptides for use within such assays includefull length lung tumor proteins and portions thereof to which thebinding agent binds, as described above.

The solid support may be any material known to those of ordinary skillin the art to which the tumor protein may be attached. For example, thesolid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681. The binding agent may beimmobilized on the solid support using a variety of techniques known tothose of skill in the art, which are amply described in the patent andscientific literature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the agent and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In general, contacting a well of a plasticmicrotiter plate (such as polystyrene or polyvinylchloride) with anamount of binding agent ranging from about 10 ng to about 10 μg, andpreferably about 100 ng to about 1 μg, is sufficient to immobilize anadequate amount of binding agent.

Covalent attachment of binding agent to a solid support may generally beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the binding agent. For example, the bindingagent may be covalently attached to supports having an appropriatepolymer coating using benzoquinone or by condensation of an aldehydegroup on the support with an amine and an active hydrogen on the bindingpartner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991,at A12-A13).

In certain embodiments, the assay is a two-antibody sandwich assay. Thisassay may be performed by first contacting an antibody that has beenimmobilized on a solid support, commonly the well of a microtiter plate,with the sample, such that polypeptides within the sample are allowed tobind to the immobilized antibody. Unbound sample is then removed fromthe immobilized polypeptide-antibody complexes and a detection reagent(preferably a second antibody capable of binding to a different site onthe polypeptide) containing a reporter group is added. The amount ofdetection reagent that remains bound to the solid support is thendetermined using a method appropriate for the specific reporter group.

More specifically, once the antibody is immobilized on the support asdescribed above, the remaining protein binding sites on the support aretypically blocked. Any suitable blocking agent known to those ofordinary skill in the art, such as bovine serum albumin or Tween 20™(Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is thenincubated with the sample, and polypeptide is allowed to bind to theantibody. The sample may be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is a period of timethat is sufficient to detect the presence of polypeptide within a sampleobtained from an individual with lung cancer. Preferably, the contacttime is sufficient to achieve a level of binding that is at least about95% of that achieved at equilibrium between bound and unboundpolypeptide. Those of ordinary skill in the art will recognize that thetime necessary to achieve equilibrium may be readily determined byassaying the level of binding that occurs over a period of time. At roomtemperature, an incubation time of about 30 minutes is generallysufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20™. The secondantibody, which contains a reporter group, may then be added to thesolid support. Preferred reporter groups include those groups recitedabove.

The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound polypeptide. An appropriate amount of time may generally bedetermined by assaying the level of binding that occurs over a period oftime. Unbound detection reagent is then removed and bound detectionreagent is detected using the reporter group. The method employed fordetecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of a cancer, such as lung cancer,the signal detected from the reporter group that remains bound to thesolid support is generally compared to a signal that corresponds to apredetermined cut-off value. In one preferred embodiment, the cut-offvalue for the detection of a cancer is the average mean signal obtainedwhen the immobilized antibody is incubated with samples from patientswithout the cancer. In general, a sample generating a signal that isthree standard deviations above the predetermined cut-off value isconsidered positive for the cancer. In an alternate preferredembodiment, the cut-off value is determined using a Receiver OperatorCurve, according to the method of Sackett et al., Clinical Epidemiology:A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p.106-7. Briefly, in this embodiment, the cut-off value may be determinedfrom a plot of pairs of true positive rates (i.e., sensitivity) andfalse positive rates (100%-specificity) that correspond to each possiblecut-off value for the diagnostic test result. The cut-off value on theplot that is the closest to the upper left-hand corner (i.e., the valuethat encloses the largest area) is the most accurate cut-off value, anda sample generating a signal that is higher than the cut-off valuedetermined by this method may be considered positive. Alternatively, thecut-off value may be shifted to the left along the plot, to minimize thefalse positive rate, or to the right, to minimize the false negativerate. In general, a sample generating a signal that is higher than thecut-off value determined by this method is considered positive for acancer.

In a related embodiment, the assay is performed in a flow-through orstrip test format, wherein the binding agent is immobilized on amembrane, such as nitrocellulose. In the flow-through test, polypeptideswithin the sample bind to the immobilized binding agent as the samplepasses through the membrane. A second, labeled binding agent then bindsto the binding agent-polypeptide complex as a solution containing thesecond binding agent flows through the membrane. The detection of boundsecond binding agent may then be performed as described above. In thestrip test format, one end of the membrane to which binding agent isbound is immersed in a solution containing the sample. The samplemigrates along the membrane through a region containing second bindingagent and to the area of immobilized binding agent. Concentration ofsecond binding agent at the area of immobilized antibody indicates thepresence of a cancer. Typically, the concentration of second bindingagent at that site generates a pattern, such as a line, that can be readvisually. The absence of such a pattern indicates a negative result. Ingeneral, the amount of binding agent immobilized on the membrane isselected to generate a visually discernible pattern when the biologicalsample contains a level of polypeptide that would be sufficient togenerate a positive signal in the two-antibody sandwich assay, in theformat discussed above. Preferred binding agents for use in such assaysare antibodies and antigen-binding fragments thereof. Preferably, theamount of antibody immobilized on the membrane ranges from about 25 ngto about 1 g, and more preferably from about 50 ng to about 500 ng. Suchtests can typically be performed with a very small amount of biologicalsample.

Of course, numerous other assay protocols exist that are suitable foruse with the tumor proteins or binding agents of the present invention.The above descriptions are intended to be exemplary only. For example,it will be apparent to those of ordinary skill in the art that the aboveprotocols may be readily modified to use lung tumor polypeptides todetect antibodies that bind to such polypeptides in a biological sample.The detection of such lung tumor protein specific antibodies maycorrelate with the presence of a cancer.

A cancer may also, or alternatively, be detected based on the presenceof T cells that specifically react with a lung tumor protein in abiological sample. Within certain methods, a biological samplecomprising CD4⁺ and/or CD8⁺ T cells isolated from a patient is incubatedwith a lung tumor polypeptide, a polynucleotide encoding such apolypeptide and/or an APC that expresses at least an immunogenic portionof such a polypeptide, and the presence or absence of specificactivation of the T cells is detected. Suitable biological samplesinclude, but are not limited to, isolated T cells. For example, T cellsmay be isolated from a patient by routine techniques (such as byFicoll/Hypaque density gradient centrifugation of peripheral bloodlymphocytes). T cells may be incubated in vitro for 2-9 days (typically4 days) at 37° C. with polypeptide (e.g., 5-25 μg/ml). It may bedesirable to incubate another aliquot of a T cell sample in the absenceof lung tumor polypeptide to serve as a control. For CD4⁺ T cells,activation is preferably detected by evaluating proliferation of the Tcells. For CD8⁺ T cells, activation is preferably detected by evaluatingcytolytic activity. A level of proliferation that is at least two foldgreater and/or a level of cytolytic activity that is at least 20%greater than in disease-free patients indicates the presence of a cancerin the patient.

As noted above, a cancer may also, or alternatively, be detected basedon the level of mRNA encoding a lung tumor protein in a biologicalsample. For example, at least two oligonucleotide primers may beemployed in a polymerase chain reaction (PCR) based assay to amplify aportion of a lung tumor cDNA derived from a biological sample, whereinat least one of the oligonucleotide primers is specific for (i.e.,hybridizes to) a polynucleotide encoding the lung tumor protein. Theamplified cDNA is then separated and detected using techniques wellknown in the art, such as gel electrophoresis. Similarly,oligonucleotide probes that specifically hybridize to a polynucleotideencoding a lung tumor protein may be used in a hybridization assay todetect the presence of polynucleotide encoding the tumor protein in abiological sample.

To permit hybridization under assay conditions, oligonucleotide primersand probes should comprise an oligonucleotide sequence that has at leastabout 60%, preferably at least about 75% and more preferably at leastabout 90%, identity to a portion of a polynucleotide encoding a lungtumor protein that is at least 10 nucleotides, and preferably at least20 nucleotides, in length. Preferably, oligonucleotide primers and/orprobes hybridize to a polynucleotide encoding a polypeptide describedherein under moderately stringent conditions, as defined above.Oligonucleotide primers and/or probes which may be usefully employed inthe diagnostic methods described herein preferably are at least 10-40nucleotides in length. In a preferred embodiment, the oligonucleotideprimers comprise at least 10 contiguous nucleotides, more preferably atleast 15 contiguous nucleotides, of a DNA molecule having a sequencerecited herein. Techniques for both PCR based assays and hybridizationassays are well known in the art (see, for example, Mullis et al., ColdSpring Harbor Symp. Quant. Biol., 51:263, 1987; Erlich ed., PCRTechnology, Stockton Press, N.Y., 1989).

One preferred assay employs RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma biological sample, such as biopsy tissue, and is reverse transcribedto produce cDNA molecules. PCR amplification using at least one specificprimer generates a cDNA molecule, which may be separated and visualizedusing, for example, gel electrophoresis. Amplification may be performedon biological samples taken from a test patient and from an individualwho is not afflicted with a cancer. The amplification reaction may beperformed on several dilutions of cDNA spanning two orders of magnitude.A two-fold or greater increase in expression in several dilutions of thetest patient sample as compared to the same dilutions of thenon-cancerous sample is typically considered positive.

In another embodiment, the compositions described herein may be used asmarkers for the progression of cancer. In this embodiment, assays asdescribed above for the diagnosis of a cancer may be performed overtime, and the change in the level of reactive polypeptide(s) orpolynucleotide(s) evaluated. For example, the assays may be performedevery 24-72 hours for a period of 6 months to 1 year, and thereafterperformed as needed. In general, a cancer is progressing in thosepatients in whom the level of polypeptide or polynucleotide detectedincreases over time. In contrast, the cancer is not progressing when thelevel of reactive polypeptide or polynucleotide either remains constantor decreases with time.

Certain in vivo diagnostic assays may be performed directly on a tumor.One such assay involves contacting tumor cells with a binding agent. Thebound binding agent may then be detected directly or indirectly via areporter group. Such binding agents may also be used in histologicalapplications. Alternatively, polynucleotide probes may be used withinsuch applications.

As noted above, to improve sensitivity, multiple lung tumor proteinmarkers may be assayed within a given sample. It will be apparent thatbinding agents specific for different proteins provided herein may becombined within a single assay. Further, multiple primers or probes maybe used concurrently. The selection of tumor protein markers may bebased on routine experiments to determine combinations that results inoptimal sensitivity. In addition, or alternatively, assays for tumorproteins provided herein may be combined with assays for other knowntumor antigens.

DIAGNOSTIC KITS

The present invention further provides kits for use within any of theabove diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to a lung tumor protein. Such antibodiesor fragments may be provided attached to a support material, asdescribed above. One or more additional containers may enclose elements,such as reagents or buffers, to be used in the assay. Such kits mayalso, or alternatively, contain a detection reagent as described abovethat contains a reporter group suitable for direct or indirect detectionof antibody binding.

Alternatively, a kit may be designed to detect the level of mRNAencoding a lung tumor protein in a biological sample. Such kitsgenerally comprise at least one oligonucleotide probe or primer, asdescribed above, that hybridizes to a polynucleotide encoding a lungtumor protein. Such an oligonucleotide may be used, for example, withina PCR or hybridization assay. Additional components that may be presentwithin such kits include a second oligonucleotide and/or a diagnosticreagent or container to facilitate the detection of a polynucleotideencoding a lung tumor protein.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 IDENTIFICATION AND CHARACTERIZATION OF LUNG TUMOR PROTEINcDNAS

This Example illustrates the identification of cDNA molecules encodinglung tumor proteins.

A. Isolation of cDNA Sequences from Lung Adenocarcinoma Libraries usingConventional cDNA Library Subtraction

A human lung adenocarcinoma cDNA expression library was constructed frompoly A⁺ RNA from patient tissues (# 40031486) using a SuperscriptPlasmid System for cDNA Synthesis and Plasmid Cloning kit (BRL LifeTechnologies, Gaithersburg, Md.) following the manufacturer's protocol.Specifically, lung carcinoma tissues were homogenized with polytron(Kinematica, Switzerland) and total RNA was extracted using Trizolreagent (BRL Life Technologies) as directed by the manufacturer. Thepoly A⁺ RNA was then purified using an oligo dT cellulose column asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989.First-strand cDNA was synthesized using the NotI/Oligo-dT18 primer.Double-stranded cDNA was synthesized, ligated with BstXI/EcoRI adaptors(Invitrogen, San Diego, Calif.) and digested with NotI. Following sizefractionation with cDNA size fractionation columns (BRL LifeTechnologies), the cDNA was ligated into the BstXI/NotI site of pcDNA3.1(Invitrogen) and transformed into ElectroMax E. coli DH10B cells (BRLLife Technologies) by electroporation. A total of 3×10⁶ independentcolonies were generated.

Using the same procedure, a normal human cDNA expression library wasprepared from a panel of normal tissue specimens, including lung, liver,pancreas, skin, kidney, brain and resting PBMC.

cDNA library subtraction was performed using the above lungadenocarcinoma and normal tissue cDNA libraries, as described by Hara etal. (Blood, 84:189-199, 1994) with some modifications. Specifically, alung adenocarcinoma-specific subtracted cDNA library was generated asfollows. The normal tissue cDNA library (80 μg) was digested with BamHIand XhoI, followed by a filling-in reaction with DNA polymerase Klenowfragment. After phenol-chloroform extraction and ethanol precipitation,the DNA was dissolved in 133 μl of H₂O, heat-denatured and mixed with133 μl (133 μg) of Photoprobe biotin (Vector Laboratories, Burlingame,Calif.). As recommended by the manufacturer, the resulting mixture wasirradiated with a 270 W sunlamp on ice for 20 minutes. AdditionalPhotoprobe biotin (67 μl) was added and the biotinylation reaction wasrepeated. After extraction with butanol five times, the DNA wasethanol-precipitated and dissolved in 23 μl H₂O. The resulting DNA, plusother highly redundant cDNA clones that were frequently recovered inprevious lung subtractions formed the driver DNA.

To form the tracer DNA, 10 μg lung adenocarcinoma cDNA library wasdigested with NotI and SpeI, phenol chloroform extracted and passedthrough Chroma spin-400 columns (Clontech, Palo Alto, Calif.).Typically, 5 μg of cDNA was recovered after the sizing column. Followingethanol precipitation, the tracer DNA was dissolved in 5 μl H₂O. TracerDNA was mixed with 15 μl driver DNA and 20 μl of 2×hybridization buffer(1.5 M NaCl/10 mM EDTA/50 mM HEPES pH 7.5/0.2% sodium dodecyl sulfate),overlaid with mineral oil, and heat-denatured completely. The sample wasimmediately transferred into a 68° C. water bath and incubated for 20hours (long hybridization [LH]). The reaction mixture was then subjectedto a streptavidin treatment followed by phenol/chloroform extraction.This process was repeated three more times. Subtracted DNA wasprecipitated, dissolved in 12 μl H₂O, mixed with 8 μl driver DNA and 20μl of 2×hybridization buffer, and subjected to a hybridization at 68° C.for 2 hours (short hybridization [SH]). After removal of biotinylateddouble-stranded DNA, subtracted cDNA was ligated into NotI/SpeI site ofchloramphenicol resistant pBCSK⁺ (Stratagene, La Jolla, Calif.) andtransformed into ElectroMax E. Coli DH10B cells by electroporation togenerate a lung adenocarcinoma specific subtracted cDNA library,referred to as LAT-S1 Similarly, LAT-S2 was generated by including 23genes that were over-expressed in the tracer as additional drivers.

A second human lung adenocarcinoma cDNA expression library wasconstructed using adenocarcinoma tissue from a second patient (# 86-66)and used to prepare a second lung adenocarcinoma-specific subtractedcDNA library (referred to as LAT2-S2), as described above, using thesame panel of normal tissues and the additional genes over-expressed inLAT-S1.

A third human metastatic lung adenocarcinoma library was constructedfrom a pool of two lung pleural effusions with lung and gastricadenocarcinoma origins. The subtracted cDNA library, Mets-sub2 wasgenerated as described above using the same panel of normal tissues.However, the Mets-sub3 subtracted library was constructed by including51 additional genes as drivers. These 51 genes were recovered inMets-sub2, representing over-expressed housekeeping genes in thetesters. As a result, Mets-sub3 is more complexed and normalized.

A total of 16 cDNA fragments isolated from LAT-S1, 585 cDNA fragmentsisolated from LAT-S2, 568 cDNA clones from LAT2-S2, 15 cDNA clones fromMets-sub2 and 343 cDNA clones from Mets-sub3, described above, werecolony PCR amplified and their mRNA expression levels in lung tumor,normal lung, and various other normal and tumor tissues were determinedusing microarray technology (Incyte, Palo Alto, Calif.). Briefly, thePCR amplification products were dotted onto slides in an array format,with each product occupying a unique location in the array. mRNA wasextracted from the tissue sample to be tested, reverse transcribed, andfluorescent-labeled cDNA probes were generated. The microarrays wereprobed with the labeled cDNA probes, the slides scanned and fluorescenceintensity was measured. This intensity correlates with the hybridizationintensity. Seventy-three non-redundant cDNA clones, of which 42 werefound to be unique, showed over-expression in lung tumors, withexpression in normal tissues tested (lung, skin, lymph node, colon,liver, pancreas, breast, heart, bone marrow, large intestine, kidney,stomach, brain, small intestine, bladder and salivary gland) beingeither undetectable, or at significantly lower levels compared to lungadenocarcinoma tumors. These clones were further characterized by DNAsequencing with a Perkin Elmer/Applied Biosystems Division AutomatedSequencer Model 373A and/or Model 377 (Foster City, Calif.).

The sequences were compared to known sequences in the gene bank usingthe EMBL GenBank databases (release 96). No significant homologies werefound to the sequence provided in SEQ ID NO: 67, with no apparenthomology to previously identified expressed sequence tags (ESTs). Thesequences of SEQ ID NO: 60, 62, 65, 66, 69-71, 74, 76, 79, 80, 84, 86,89-92, 95, 97 and 98 were found to show some homology to previouslyidentified expressed sequence tags (ESTs). The cDNA sequences of SEQ IDNO: 59, 61, 63, 64, 67, 68, 72, 73, 75, 77, 78, 81-83, 85, 87, 88, 93,94, 96, 99 and 100 showed homology to previously identified genes. Thefull-length cDNA sequences for the clones of SEQ ID NO: 96 and 100 areprovided in SEQ ID NO: 316 and 318, respectively. The amino acidsequences for the clones of SEQ ID NO: 59, 61, 63, 64, 68, 73, 82, 83,94, 96 and 100 are provided in SEQ ID NO: 331, 328, 329, 332, 327, 333,330, 326, 325, 324 and 335, respectively. A predicted amino acidsequence encoded by the sequence of SEQ ID NO: 69 (referred to as L552S)is provided in SEQ ID NO: 786.

Further studies led to the isolation of an extended cDNA sequence, andopen reading frame, for L552S (SEQ ID NO: 790). The predicted amino acidsequence encoded by the cDNA sequence of SEQ ID NO: 790 is provided inSEQ ID NO: 791. The determined cDNA sequence of an isoform of L552S isprovided in SEQ ID NO: 792, with the corresponding predicted amino acidsequence being provided in SEQ ID NO: 793. Subsequent studies led to theisolation of the full-length cDNA sequence of L552S (SEQ ID NO: 808).The corresponding amino acid sequence is provided in SEQ ID NO: 809. Nohomologies were found to the protein sequence of L552S. However,nucleotides 533-769 of the full-length cDNA sequence were found to showhomology to a previously identified DNA sequence.

Full-length cloning efforts on L552S led to the isolation of threeadditional cDNA sequences (SEQ ID NO: 810-812) from a metastatic lungadenocarcinoma library. The sequence of SEQ ID NO: 810 was found to showsome homology to previously identified human DNA sequences. The sequenceof SEQ ID NO: 811 was found to show some homology to a previouslyidentified DNA sequence. The sequence of SEQ ID NO: 812 was found toshow some homology to previously identified ESTs.

The gene of SEQ ID NO: 84 (referred to as L551S) was determined byreal-time RT-PCR analysis to be over-expressed in 2/9 primaryadenocarcinomas and to be expressed at lower levels in 2/2 metastaticadenocarcinomas and 1/2 squamous cell carcinomas. No expression wasobserved in normal tissues, with the exception of very low expression innormal stomach. Further studies on L551S led to the isolation of the 5′and 3′ cDNA consensus sequences provided in SEQ ID NO: 801 and 802,respectively. The L551S 5′ sequence was found to show some homology tothe previously identified gene STY8 (cDNA sequence provided in SEQ IDNO: 803; corresponding amino acid sequence provided in SEQ ID NO: 805),which is a mitogen activated protein kinase phosphatase. However, nosignificant homologies were found to the 3′ sequence of L551S.Subsequently, an extended cDNA sequence for L551S was isolated (SEQ IDNO: 804). The corresponding amino acid sequence is provided in SEQ IDNO: 806. Further studies led to the isolation of two independentfull-length clones for L551S (referred to as 54298 and 54305). These twoclones have five nucleotide differences compared to the STY8 DNAsequence. Two of these differences are single nucleotide polymorphismswhich do not effect the encoded amino acid sequences. The other threenucleotide differences are consistent between the two L551S clones butlead to encoded amino acid sequences that are different from the STY8protein sequence. The determined cDNA sequences for the L551Sfull-length clones 54305 and 54298 are provided in SEQ ID NO: 825 and826, respectively, with the amino acid sequence for L551S being providedin SEQ ID NO: 827.

B. Isolation of cDNA Sequences from Lung Adenocarcinoma Libraries usingPCR-Based cDNA Library Subtraction

cDNA clones from a PCR-based subtraction library, containing cDNA from apool of two human lung primary adenocarcinomas subtracted against a poolof nine normal human tissue cDNAs including skin, colon, lung,esophagus, brain, kidney, spleen, pancreas and liver, (Clontech, PaloAlto, Calif.) were derived and submitted to a first round of PCRamplification. This library (referred to as ALT-1) was subjected to asecond round of PCR amplification, following the manufacturer'sprotocol. The expression levels of 760 cDNA clones in lung tumor, normallung, and various other normal and tumor tissues, were examined usingmicroarray technology as described above. A total of 118 clones, ofwhich 55 were unique, were found to be over-expressed in lung tumortissue, with expression in normal tissues tested (lung, skin, lymphnode, colon, liver, pancreas, breast, heart, bone marrow, largeintestine, kidney, stomach, brain, small intestine, bladder and salivarygland) being either undetectable, or at significantly lower levels. Thesequences were compared to known sequences in the gene bank using theEMBL and GenBank databases (release 96). No significant homologies(including ESTs) were found to the sequence provided in SEQ ID NO: 44.The sequences of SEQ ID NO: 1, 11, 13, 15, 20, 23-27, 29, 30, 33, 34,39, 41, 43, 45, 46, 51 and 57 were found to show some homology topreviously identified expressed sequence tags (ESTs). The cDNA sequencesof SEQ ID NO: 2-10, 12, 14, 16-19, 21, 22, 28, 31, 32, 35-38, 40, 42,44, 47-50, 52-56 and 58 showed homology to previously identified genes.The full-length cDNA sequences for the clones of SEQ ID NO: 18, 22, 31,35, 36 and 42 are provided in SEQ ID NO: 320, 319, 323, 321, 317, 321and 322, respectively, with the corresponding amino acid sequences beingprovided in SEQ ID NO: 337, 336, 340, 338, 334, and 339, respectively.

Further studies led to the isolation of an extended cDNA sequence forthe clone of SEQ ID NO: 33 (referred to as L801P). This extended cDNAsequence (provided in SEQ ID NO: 796), was found to contain threepotential open reading frames (ORFs). The predicted amino acid sequencesencoded by these three ORFs are provided in SEQ ID NO: 797-799,respectively. Additional full-length cloning efforts led to stillfurther extended cDNA sequence for L801P, set forth in SEQ ID NO:1669,in addition to five potential open reading frames (ORFs 4-9; SEQ ID NOs:1670-1675, respectively) encoded by the extended cDNA sequence.Moreover, L801P was mapped to chromosomal region 20p13 and a 137 aminoacid ORF from this genomic region was identified that corresponds toORF4 (SEQ ID NO: 1670), suggesting that this is likely an authentic ORFfor L801P.

By microarray analysis, L801P was overexpressed by 2-fold or greater inthe lung tumor probe groups compared to the normal tissue probe group(not shown). By real-time PCR analysis, greater than 50% of lungadenocarcinoma and greater than 30% of lung squamous cell carcinomatumor samples tested had elevated L801P expression relative to normallung tissue. Of those that displayed elevated L801P, the level ofexpression was greater than 10-fold higher than in normal lung tissuesamples. Moreover, low or no expression of L801P was detected in anextensive panel of normal tissue RNAs.

We have also found that L801P expression is detected in a number ofother tumor types, including breast, prostate, ovarian and colon tumors,and thus may have diagnostic and/or therapeutic utility in these cancertypes as well.

In subsequent studies, a full-length cDNA sequence for the clone of SEQID NO: 44 (referred to as L844P) was isolated (provided in SEQ ID NO:800). Comparison of this sequence with those in the public databasesrevealed that the 470 bases at the 5′ end of the sequence show homologyto the known gene dihydrodiol dehydrogenase, thus indicating that L844Pis a novel transcript of the dihydrodiol dehydrogenase family having2007 base pairs of previously unidentified 3′ untranslated region.

The predicted amino acid sequence encoded by the sequence of SEQ ID NO:46 (referred to as L840P) is provided in SEQ ID NO: 787. An extendedcDNA sequence for L840P, which was determined to include an open readingframe, is provided in SEQ ID NO: 794. The predicted amino acid sequenceencoded by the cDNA sequence of SEQ ID NO: 794 is provided in SEQ ID NO:795. The full-length cDNA sequence for the clone of SEQ ID NO: 54(referred to as L548S) is provided in SEQ ID NO: 788, with thecorresponding amino acid sequence being provided in SEQ ID NO: 789.

Northern blot analyses of the genes of SEQ ID NO: 25 and 46 (referred toas L839P and L840P, respectively) were remarkably similar. Both geneswere expressed in 1/2 lung adenocarcinomas as two bands of 3.6 kb and1.6 kb. No expression of L839P was observed in normal lung or trachea.No expression of L840P was observed in normal bone marrow, resting oractivated PBMC, esophagus, or normal lung. Given the similar expressionpatterns, L839P and L840P may be derived from the same gene.

Further studies on L773P (SEQ ID NO: 58) resulted in the isolation ofthe extended consensus cDNA sequence provided in SEQ ID NO: 807.

Additional lung adenocarcinoma cDNA clones were isolated as follows. AcDNA library was prepared from a pool of two lung adenocarcinomas andsubtracted against cDNA from a panel of normal tissues including lung,brain, liver, kidney, pancreas, skin, heart and spleen. The subtractionwas performed using a PCR-based protocol (Clontech), which was modifiedto generate larger fragments. Within this protocol, tester and driverdouble stranded cDNA were separately digested with five restrictionenzymes that recognize six-nucleotide restriction sites (MluI, MscI,PvuII, SalI and StuI). This digestion resulted in an average cDNA sizeof 600 bp, rather than the average size of 300 bp that results fromdigestion with RsaI according to the Clontech protocol. The ends of therestriction digested tester cDNA were filled in to generate blunt endsfor adapter ligation. This modification did not affect the subtractionefficiency. Two tester populations were then created with differentadapters, and the driver library remained without adapters. The testerand driver libraries were then hybridized using excess driver cDNA. Inthe first hybridization step, driver was separately hybridized with eachof the two tester cDNA populations. This resulted in populations of (a)unhybridized tester cDNAs, (b) tester cDNAs hybridized to other testercDNAs, (c) tester cDNAs hybridized to driver cDNAs and (d) unhybridizeddriver cDNAs. The two separate hybridization reactions were thencombined, and rehybridized in the presence of additional denatureddriver cDNA. Following this second hybridization, in addition topopulations (a) through (d), a fifth population (e) was generated inwhich tester cDNA with one adapter hybridized to tester cDNA with thesecond adapter. Accordingly, the second hybridization step resulted inenrichment of differentially expressed sequences which could be used astemplates for PCR amplification with adaptor-specific primers.

The ends were then filled in, and PCR amplification was performed usingadaptor-specific primers. Only population (e), which contained testercDNA that did not hybridize to driver cDNA, was amplified exponentially.A second PCR amplification step was then performed, to reduce backgroundand further enrich differentially expressed sequences.

Fifty-seven cDNA clones were isolated from the subtracted library(referred to as LAP1) and sequenced. The determined cDNA sequences for16 of these clones are provided in SEQ ID NO: 101-116. The sequences ofSEQ ID NO: 101 and 114 showed no significant homologies to previouslyidentified sequences. The sequences of SEQ ID NO: 102-109 and 112 showedsome similarity to previously identified sequences, while the sequencesof SEQ ID NO: 113, 115 and 116 showed some similarity to previouslyisolated ESTs.

An additional 502 clones analyzed from the LAP1 library were sequencedand the determined cDNA sequences are shown in SEQ ID NO:828-1239 and1564-1653.

C. Isolation of cDNA Sequences from Small Cell Lung Carcinoma Librariesusing PCR-Based cDNA Library Subtraction

A subtracted cDNA library for small cell lung carcinoma (referred to asSCL1) was prepared using essentially the modified PCR-based subtractionprocess described above. cDNA from small cell lung carcinoma wassubtracted against cDNA from a panel of normal tissues, including normallung, brain, kidney, liver, pancreas, skin, heart, lymph node andspleen. Both tester and driver poly A+ RNA were initially amplifiedusing SMART PCR cDNA synthesis kit (Clontech, Palo Alto, Calif.). Thetester and driver double stranded cDNA were separately digested withfive restriction enzymes (DraI, MscI, PvuII, SmaI, and StuI). Theserestriction enzymes generated blunt end cuts and the digestion resultedin an average insert size of 600 bp. Digestion with this set ofrestriction enzymes eliminates the step required to generate blunt endsby filling in of the cDNA ends. These modifications did not affectsubtraction efficiency.

Eighty-five clones were isolated and sequenced. The determined cDNAsequences for 31 of these clones are provided in SEQ ID NO: 117-147. Thesequences of SEQ ID NO: 122, 124, 126, 127, 130, 131, 133, 136, 139 and147 showed no significant homologies to previously identified sequences.The sequences of SEQ ID NO: 120, 129, 135, 137, 140, 142, 144 and 145showed some similarity to previously identified gene sequences, whilethe sequences of SEQ ID NO: 114, 118, 119, 121, 123, 125, 128, 132, 134,138, 141, 143 and 147 showed some similarity to previously isolatedESTs.

In further studies, three additional cDNA libraries were generated frompoly A+ RNA from a single small cell lung carcinoma sample subtractedagainst a pool of poly A+ RNA from nine normal tissues (lung, brain,kidney, liver, pancreas, skin, heart pituitary gland and spleen). Forthe first library (referred to as SCL2), the subtraction was carried outessentially as described above for the LAP1 library, with the exceptionthat the tester and driver were digested with PvuII, StuI, MscI andDraI. The ratio of tester and driver cDNA used was as recommended byClontech. For the second library (referred to as SCL3), subtraction wasperformed essentially as for SCL2 except that cDNA for highly redundantclones identified from the SCL2 library was included in the driver cDNA.Construction of the SCL4 library was performed essentially as describedfor the SCL3 library except that a higher ratio of driver to tester wasemployed.

Each library was characterized by DNA sequencing and database analyses.The determined cDNA sequence for 35 clones isolated from the SCL2library are provided in SEQ ID NO: 245-279, with the determined cDNAsequences for 21 clones isolated from the SCL3 library and for 15 clonesisolated from the SCL4 library being provided in SEQ ID NO: 280-300 and301-315, respectively. The sequences of SEQ ID NO: 246, 254, 261, 262,304, 309 and 311 showed no significant homologies to previouslyidentified sequences. The sequence of SEQ ID NO: 245, 248, 255, 266,270, 275, 280, 282, 283, 288-290, 292, 295, 301 and 303 showed somehomology to previously isolated ESTs, while the sequences of SEQ ID NO:247, 249-253, 256-260, 263-265, 267-269, 271-274, 276-279, 281, 284-287,291, 293, 294, 296-300, 302, 305-308, 310 and 312-315 showed somehomology to previously identified gene sequences.

3264 cDNA clones from three PCR-based subtracted cDNA libraries wereanalyzed by cDNA microarray technology as part of Lung Chip 5. Of the3264 cDNA clones 960 clones came from SQL1 library, 768 clones came fromSCL1 library, and 1536 clones came from SCL3 and SCL4 libraries. 35pairs of fluorescent labeled cDNA probes were used for the microarrayanalysis. Each probe pair included a lung tumor probe paired with anormal tissue probe. The expression data was analyzed. 498 cDNA cloneswere found to be overexpressed by 2-fold or greater in the small celland/or non-small cell lung tumor probe groups compared to the normaltissue probe group. Also, the mean expression values for these clones innormal tissues were below 0.1 (range of expression is from 0.001 to 10).The cDNA sequences disclosed in SEQ ID NO:1240-1563 represent 324non-redundant clones.

The following sequences were novel based on database analysis includingGenBank and GeneSeq: SEQ ID NO:1240, 1243, 1247, 1269, 1272, 1280, 1283,1285, 1286, 1289, 1300, 1309, 1318, 1319, 1327, 1335, 1339, 1346, 1359,1369, 1370, 1371, 1393, 1398, 1405, 1408, 1413, 1414, 1417, 1422, 1429,1432, 1435, 1436, 1438-1442, 1447, 1450, 1453, 1463, 1467, 1470, 1473,1475, 1482, 1486, 1491-1494, 1501, 1505, 1506, 1514-1517, 1520, 1522,1524, 1535, 1538, 1542, 1543, 1547, 1554, 1557, 1559, 1561, and 1563.

Full-length sequence for contig 139 (SEQ ID NO: 1467), also known asL985P, was identified by searching public databases using SEQ ID NO:1467 as a query. By this approach, L985 was identified as cell surfaceimmunomodulator-2 (CSIMM-2), the cDNA sequence of which is set forth inSEQ ID NO: 1676, encoding a protein having the sequence set forth in SEQID NO: 1677.

By microarray analysis, L985P was overexpressed by 2-fold or greater inthe lung tumor probe groups compared to the normal tissue probe group.Moreover, the mean expression values for L985P in normal tissues wasbelow 0.2 (range of expression was from 0.01 to 10). By real-time PCRanalysis, greater than 40% of small cell lung carcinoma lung tumorsamples tested had elevated L985P expression relative to normal lungtissue. Of those that displayed elevated L985P, the level of expressionwas greater than 3-fold higher than in normal lung tissue samples. Lowor no expression of L985P was detected in an extensive panel of normaltissue RNAs. These findings for L985P support its use both as adiagnositic marker for detecting the presence of lung cancer in apatient and/or as a immunotherapeutic target for the treatment of lungcancer.

D. Isolation of cDNA Sequences from a Neuroendocrine Library usingPCR-Based cDNA Library Subtraction

Using the modified PCR-based subtraction process, essentially asdescribed above for the LAP1 subtracted library, a subtracted cDNAlibrary (referred to as MLN1) was derived from a lung neuroendocrinecarcinoma that had metastasized to the subcarinal lymph node, bysubtraction with a panel of nine normal tissues, including normal lung,brain, kidney, liver, pancreas, skin, heart, lymph node and spleen.

Ninety-one individual clones were isolated and sequenced. The determinedcDNA sequences for 58 of these clones are provided in SEQ ID NO:147-222. The sequences of SEQ ID NO: 150, 151, 154, 157, 158, 159, 160,163, 174, 175, 178, 186-190, 192, 193, 195-200, 208-210, 212-215 and 220showed no significant homologies to previously identified sequences. Thesequences of SEQ ID NO: 152, 155, 156, 161, 165, 166, 176, 179, 182,184, 185, 191, 194, 221 and 222 showed some similarity to previouslyidentified gene sequences, while the sequences of SEQ ID NO: 148, 149,153, 164, 167-173, 177, 180, 181, 183, 201-207, 211 and 216-219 showedsome similarity to previously isolated ESTs.

The determined cDNA sequences of an additional 442 clones isolated fromthe MLN1 library are provided in SEQ ID NO: 341-782. The determined cDNAsequences of an additional 11 clones isolated from the MLN1 library areprovided in SEQ ID NO:1654-1664.

E. Isolation of cDNA Sequences from a Squamous Cell Lung CarcinomaLibrary using PCR-Based cDNA Library Subtraction

A subtracted cDNA library for squamous cell lung carcinoma (referred toas SQL1) was prepared, essentially using the modified PCR-basedsubtraction process described above, except the tester and driver doublestranded cDNA were separately digested with four restriction enzymes(DraI, MscI, PvuII and StuI) cDNA from a pool of two squamous cell lungcarcinomas was subtracted against cDNA from a pool of 10 normal tissues,including normal lung, brain, kidney, liver, pancreas, skin, heart,spleen, esophagus and trachea.

Seventy-four clones were isolated and sequenced. The determined cDNAsequences for 22 of these clones are provided in SEQ ID NO: 223-244. Thesequence of SEQ ID NO: 241 showed no significant homologies topreviously identified sequences. The sequences of SEQ ID NO: 223, 225,232, 233, 235, 238, 239, 242 and 243 showed some similarity topreviously identified gene sequences, while the sequences of SEQ ID NO:224, 226-231, 234, 236, 237, 240, 241 and 244 showed some similarity topreviously isolated ESTs.

The sequences of an additional 12 clones isolated duringcharacterization of cDNA libraries prepared from lung tumor tissue areprovided in SEQ ID NO: 813-824. Comparison of these sequences with thosein the GenBank database and the GeneSeq DNA database revealed nosignificant homologies to previously identified sequences.

EXAMPLE 2 SYNTHESIS OF POLYPEPTIDES

Polypeptides may be synthesized on a Perkin Elmer/Applied BiosystemsDivision 430A peptide synthesizer using FMOC chemistry with HPTU(O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate)activation. A Gly-Cys-Gly sequence may be attached to the amino terminusof the peptide to provide a method of conjugation, binding to animmobilized surface, or labeling of the peptide. Cleavage of thepeptides from the solid support may be carried out using the followingcleavage mixture: trifluoroaceticacid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleavingfor 2 hours, the peptides may be precipitated in coldmethyl-t-butyl-ether. The peptide pellets may then be dissolved in watercontaining 0.1% trifluoroacetic acid (TFA) and lyophilized prior topurification by C18 reverse phase HPLC. A gradient of 0%-60%acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA) may beused to elute the peptides. Following lyophilization of the purefractions, the peptides may be characterized using electrospray or othertypes of mass spectrometry and by amino acid analysis.

EXAMPLE 3 PREPARATION OF ANTIBODIES AGAINST LUNG CANCER ANTIGENS

Polyclonal antibodies against the lung cancer antigen L773P (SEQ ID NO:783) were prepared as follows.

Rabbits were immunized with recombinant protein expressed in andpurified from E. Coli as described above. For the initial immunization,400 μg of antigen combined with muramyl dipeptide (MDP) was injectedsubcutaneously (S.C.). Animals were boosted S.C. 4 weeks later with 200μg of antigen mixed with incomplete Freund's Adjuvant (IFA). Subsequentboosts of 100 μg of antigen mixed with IFA were injected S.C. asnecessary to induce high antibody titer responses. Serun bleeds fromimmunized rabbits were tested for L773P-specific reactivity using ELISAassays with purified protein and showed strong reactivity to L733P.Polyclonal antibodies against L773P were affinity purified from hightiter polyclonal sera using purified protein attached to a solidsupport.

EXAMPLE 4 PROTEIN EXPRESSION OF LUNG TUMOR-SPECIFIC ANTIGENS

Full-length L773P (amino acids 2-364 of SEQ ID NO: 783), with a 6× HisTag, were subcloned into the pPDM expression vector and transformed intoeither BL21 CodonPlus or BL21 pLysS host cells using standardtechniques. High levels of expression were observed in both cases.Similarly, the N-terminal portion of L773P (amino acids 2-71 of SEQ IDNO: 783; referred to as L773PA), with a 6× His tag were subcloned intothe vector pPDM and transformed into BL21 CodonPlus host cells. Lowlevels of expression were observed by N-terminal sequencing. Thesequence of the expressed constructs for L773P and L773PA are providedin SEQ ID NO: 784 and 785, respectively.

EXAMPLE 5 EXPRESSION IN E. COLI OF L548S HIS TAG FUSION PROTEIN

The L548S coding region was PCR amnplified with the following primers:

Forward primer starting at amino acid 2:

PDM-433: 5′ gctaaaggtgaccccaagaaaccaaag 3′ Tm 60° C. (SEQ ID NO:1665)

Reverse primer creating a XhoI site after the stop codon:

PDM-438: 5′ ctattaactcgagggagacagataaacagtttcttta 3′ Tm 6° C. (SEQ IDNO:1666)

The PCR product was then digested with XhoI restriction enzyme, gelpurified and then cloned into pPDM His, a modified pET28 vector with aHis tag in frame, which had been digested with Eco72I and XhoIrestriction enzymes. The correct construct was confirmed by DNA sequenceanalysis and then transformned into BL21 (DE3) pLys S and BL21 (DE3)CodonPlus RIL expression hosts.

The protein sequence of expressed recombinant L548S is shown in SEQ IDNO:1667, and the DNA sequence of expressed recombinant L7548S is shownin SEQ ID NO: 1668.

EXAMPLE 6 ADDITIONAL ANALYSES OF LUNG CHIP 5 SQL1, SCL1, SCL3 AND SCL4LIBRARIES

Additional analyses were performed on lung chip 5 using a criteria ofgreater than or equal to 2-fold over-expression in tumor probe groupsversus normnal tissues and an average expression in normal tissues ofless than or equal to 0.2. This resulted in the identification of 109non-redundant clones that are over-expressed in lung carcinomas. Assummarized in the table below, 19 cDNA clones were recovered from thelung squamous cell carcinoma subtracted library SQL1, 9 cDNA clones wererecovered from the small cell lung carcinoma library SCL1 and 81 cDNAclones were recovered from the small cell lung carcinoma libraries SCL3and SCL4.

SEQ ID Mean Mean NO: Seq. Ref. Element (384) Element (96) Ratio Signal 1Signal 2 Library 1680 58456 p0003r03c13 R0001 E7 3.09 0.424 0.137 SQL11681 58458 p0003r03c10 R0001 F5 2.31 0.408 0.176 SQL1 1682 58462p0003r04c16 R0001 H8 2.22 0.257 0.116 SQL1 1683 58469 p0003r07c12 R0002F6 2.1 0.289 0.138 SQLI 1684 58470 p0003r09c21 R0003 A11 2.55 0.4930.194 SQL1 1685 58482 p0003r12c19 R0003 G10 2.16 0.36 0.167 SQLI 168658485 p0003r12c10 R0003 H5 2.48 0.273 0.11 SQL1 1687 58501 p0004r04c23R0005 G12 2.04 0.26 0.128 SQL1 1688 58502 p0004r04c03 R0005 G2 2.170.289 0.133 SQL1 1689 58505 p0004r05c23 R0006 A12 3.08 0.454 0.148 SQLI1690 58507 p0004r06c11 R0006 C6 3.22 0.49 0.152 SQL1 1691 58509p0004r07c15 R0006 E8 3.26 0.421 0.129 SQLI 1692 58512 p0004r09c03 R0007A2 3.16 0.559 0.177 SQL1 1693 58527 p0004r12c22 R0007 H11 2.03 0.2780.137 SQL1 1694 58529 p0004r14c09 R0008 C5 2.26 0.45 0.199 SQL1 169558531 p0004r16c01 R0008 G1 2.84 0.387 0.136 SQL1 1696 58537 p0005r02c08R0009 D4 2.03 0.355 0.175 SQL1 1697 58539 p0005r03c08 R0009 F4 2.34 0.420.18 SQL1 1698 58545 p0005r07c21 R0010 E11 2.96 0.361 0.122 SQL1 169959319 p0005r10c04 R0011 D2 3.1 0.478 0.154 SCL1 1700 59322 p0005r12c01R0011 G1 2.16 0.255 0.118 SCL1 1701 59348 p0006r11c12 R0015 F6 2.330.269 0.116 SCL1 1702 59350 p0006r14c13 R0016 C7 2.41 0.447 0.185 SCL11703 59363 p0007r02c16 R0017 D8 2.12 0.421 0.199 SCL1 1704 59365p0007r03c20 R0017 F10 3.07 0.584 0.19 SCL1 1705 59370 p0007r04c10 R0017H5 2.06 0.284 0.138 SCL1 1706 59373 p0007r05c23 R0018 A12 2.95 0.4720.16 SCL1 1707 59376 p0007r06c02 R0018 D1 2.13 0.246 0.116 SCLI 170861050 p0011r02c10 R0033 D5 2.23 0.306 0.137 SCL3/4 1709 61051p0011r03c23 R0033 E12 2.9 0.298 0.103 SCL3/4 1710 61052 p0011r03c08R0033 F4 2.18 0.265 0.122 SCL3/4 1711 61054 p0011r03c16 R0033 F8 2.110.415 0.197 SCL3/4 1712 61056 p0011r04c13 R0033 G7 2.73 0.314 0.115SCL3/4 1713 61057 p0011r04c10 R0033 H5 2.45 0.463 0.189 SCL3/4 171461060 p0011r05c11 R0034 A6 3.28 0.536 0.164 SCL3/4 1715 61062p0011r06c21 R0034 C11 2.73 0.526 0.192 SCL3/4 1716 61063 p0011r06c05R0034 C3 3.61 0.513 0.142 SCL3/4 1717 61064 p0011r06c04 R0034 D2 2.580.477 0.185 SCL3/4 1718 61065 p0011r06c14 R0034 D7 4.91 0.55 0.112SCL3/4 1719 61066 p0011r06c18 R0034 D9 2.38 0.285 0.12 SCL3/4 1720 61069p0011r07c16 R0034 F8 2.25 0.426 0.189 SCL3/4 1721 61070 p0011r08c21R0034 G11 2 0.234 0.117 SCL3/4 1722 61071 p0011r08c03 R0034 G2 2.760.321 0.116 SCL3/4 1723 61074 p0011r08c16 R0034 H8 3.02 0.399 0.132SCL3/4 1724 61075 p0011r09c05 R0035 A3 3.83 0.498 0.13 SCL3/4 1725 61077p0011r10c21 R0035 C11 2.12 0.306 0.144 SCL3/4 1726 61079 p0011r11c23R0035 E12 2.04 0.22 0.108 SCL3/4 1727 61080 p0011r11c15 R0035 E8 2.760.299 0.108 SCL3/4 1728 61081 p0011r11c14 R0035 F7 2.37 0.303 0.128SCL3/4 1729 61083 p0011r12c15 R0035 G8 2.29 0.351 0.153 SCL3/4 173061085 p0011r13c05 R0036 A3 2.62 0.43 0.164 SCL3/4 1731 61086 p0011r13c09R0036 A5 2.53 0.398 0.157 SCL3/4 1732 61088 p0011r14c05 R0036 C3 4.260.702 0.165 SCL3/4 1733 61090 p0011r15c07 R0036 E4 3.16 0.429 0.136SCL3/4 1734 61091 p0011r16c16 R0036 H8 3.54 0.634 0.179 SCL3/4 173561093 p0012r02c03 R0037 C2 2.2 0.265 0.121 SCL3/4 1736 61094 p0012r02c11R0037 C6 15.17 1.79 0.118 SCL3/4 1737 61096 p0012r02c08 R0037 D4 2.440.27 0.111 SCL3/4 1738 61097 p0012r02c10 R0037 D5 4.52 0.81 0.179 SCL3/41739 61099 p0012r03c02 R0037 F1 3.34 0.39 0.117 SCL3/4 1740 61100p0012r03c06 R0037 F3 2.03 0.233 0.114 SCL3/4 1741 61103 p0012r04c17R0037 G9 2.48 0.413 0.167 SCL3/4 1742 61105 p0012r05c11 R0038 A6 3.260.501 0.154 SCL3/4 1743 61106 p0012r05c08 R0038 B4 2.46 0.354 0.144SCL3/4 1744 61110 p0012r06c15 R0038 C8 2.18 0.41 0.188 SCL3/4 1745 61113p0012r07c09 R0038 E5 2.47 0.376 0.152 SCL3/4 1746 61115 p0012r07c13R0038 E7 2.57 0.483 0.188 SCL3/4 1747 61117 p0012r07c24 R0038 F12 2.180.235 0.108 SCL3/4 1748 61118 p0012r07c18 R0038 F9 4.44 0.605 0.136SCL3/4 1749 61119 p0012r08c03 R0038 G2 2.97 0.35 0.118 SCL3/4 1750 61120p0012r08c07 R0038 G4 2.23 0.323 0.144 SCL3/4 1751 61122 p0012r08c18R0038 H9 2.23 0.373 0.168 SCL3/4 1752 61125 p0012r10c17 R0039 C9 2.10.22 0.105 SCL3/4 1753 61126 p0012r10c16 R0039 D8 2.47 0.345 0.14 SCL3/41754 61130 p0012r12c12 R0039 H6 2.66 0.282 0.106 SCL3/4 1755 61133p0012r13c24 R0040 B12 2.25 0.27 0.12 SCL3/4 1756 61134 p0012r14c23 R0040C12 2.23 0.228 0.102 SCL3/4 1757 61135 p0012r14c03 R0040 C2 2.05 0.2980.146 SCL3/4 1758 61137 p0012r14c02 R0040 D1 8.63 1.463 0.17 SCL3/4 175961139 p0012r14c14 R0040 D7 2.69 0.3 0.111 SCL3/4 1760 61143 p0012r16c02R0040 H1 2.55 0.318 0.125 SCL3/4 1761 61144 p0012r16c18 R0040 H9 2.850.318 0.112 SCL3/4 1762 61148 p0013r02c19 R0041 C10 2.33 0.463 0.199SCL3/4 1763 61151 p0013r02c03 R0041 C2 2.25 0.336 0.149 SCL3/4 176461155 p0013r04c07 R0041 G4 2.13 0.366 0.171 SCL3/4 1765 61156p0013r05c05 R0042 A3 2.73 0.38 0.139 SCL3/4 1766 61159 p0013r06c24 R0042D12 4.57 0.831 0.182 SCL3/4 1767 61160 p0013r07c19 R0042 E10 8.6 1.1910.138 SCL3/4 1768 61163 p0013r07c18 R0042 F9 2.18 0.278 0.128 SCL3/41769 61167 p0013r10c12 R0043 D6 3.13 0.39 0.124 SCL3/4 1770 61172p0013r12c03 R0043 G2 2 0.396 0.198 SCL3/4 1771 61173 p0013r12c07 R0043G4 3.73 0.72 0.193 SCL3/4 1772 61176 p0013r13c04 R0044 B2 2.34 0.4460.19 SCL3/4 1773 61177 p0013r14c01 R0044 C1 3.9 0.539 0.138 SCL3/4 177461183 p0013r15c14 R0044 F7 5.49 0.959 0.175 SCL3/4 1775 61185p0013r16c24 R0044 H12 2.25 0.409 0.182 SCL3/4 1776 61188 p0014r01c07R0045 A4 2.14 0.271 0.127 SCL3/4 1777 61192 p0014r02c19 R0045 C10 2.330.321 0.138 SCL3/4 1778 61198 p0014r04c24 R0045 H12 2.3 0.321 0.14SCL3/4 1779 61201 p0014r06c22 R0046 D11 2.43 0.269 0.111 SCL3/4 178061202 p0014r06c08 R0046 D4 2.57 0.346 0.135 SCL3/4 1781 61204p0014r07c07 R0046 E4 4.27 0.516 0.121 SCL3/4 1782 61206 p0014r07c12R0046 F6 2.18 0.364 0.167 SCL3/4 1783 61210 p0015r09c02 R0051 B1 2.430.463 0.19 SCL3/4 1784 61212 p0015r10c15 R0051 C8 2.64 0.406 0.154SCL3/4 1785 61216 p0015r11c16 R0051 F8 2.28 0.278 0.122 SCL3/4 178661225 p0015r14c12 R0052 D6 2.25 0.25 0.111 SCL3/4 1787 61226 p0015r14c14R0052 D7 2.54 0.3 0.118 SCL3/4 1788 61227 p0015r16c18 R0052 H9 2.060.312 0.151 SCL3/4

The ratio of signal 1 to signal 2 in the table above provides a measureof the level of expression of the identified sequences in tumor versusnormal tissues. For example, for SEQ ID NO: 1669, the tumor-specificsignal was 3.09 times that of the signal for the normal tissues tested;for SEQ ID NO: 1670, the tumor-specific signal was 2.31 times that ofthe signal for normal tissues, etc.

EXAMPLE 7 REAL-TIME PCR ANALYSES OF LUNG TUMOR SEQUENCES

Real Time PCR was performed on a subset of the lung tumor sequencesdisclosed herein in order to further evaluate their expression profilesin various tumor and normal tissues. Briefly, quantitation of PCRproduct relies on the few cycles where the amount of DNA amplifieslogarithmically from barely above the background to the plateau. Usingcontinuous fluorescence monitoring, the threshold cycle number where DNAamplifies logarithmically is easily determined in each PCR reaction.There are two fluorescence detecting systems. One is based upon adouble-strand DNA specific binding dye SYBR Green I dye. The other usesTaqMan probe containing a Reporter dye at the 5′ end (FAM) and aQuencher dye at the 3′ end (TAMRA) (Perkin Elmer/Applied BiosystemsDivision, Foster City, Calif.). Target-specific PCR amplificationresults in cleavage and release of the Reporter dye from theQuencher-containing probe by the nuclease activity of AmpliTaq Gold™(Perkin Elmer/Applied Biosystems Division, Foster City, Calif.). Thus,fluorescence signal generated from released reporter dye is proportionalto the amount of PCR product. Both detection methods have been found togenerate comparable results To compare the relative level of geneexpression in multiple tissue samples, a panel of cDNAs is constructedusing RNA from tissues and/or cell lines, and real-time PCR is performedusing gene specific primers to quantify the copy number in each cDNAsample. Each cDNA sample is generally performed in duplicate and eachreaction repeated in duplicated plates. The final Real-time PCR resultis typically reported as an average of copy number of a gene of interestnormalized against internal actin number in each cDNA sample. Real-timePCR reactions may be performed on a GeneAmp 5700 Detector using SYBRGreen I dye or an ABI PRISM 7700 Detector using the TaqMan probe (PerkinElmer/Applied Biosystems Division, Foster City, Calif.).

Results obtained from Real-time PCR analysis of a number of lungtumor-specific sequences disclosed herein are summarized in the tablebelow. In addition, extended cDNA sequences for many of these cloneswere obtained by searching public sequence databases. The extendedsequences, and the proteins encoded by those sequences, are identifiedby SEQ ID NO: in the table below.

Extended Encoded Clone Clone SEQ cDNA Polypeptide Library Name No. IDNO: Real-Time PCR Results Sequence Sequence SQL1 L972P 47988 1789Overexpressed in 1/7 lung squamous tumor, 1/3 HN squamous tumor. Low orno expression in normal tissues. SQL1 L979P 48005 1790 Over-expressed in2/7 squamous lung 1791 1806 tumors, 1/3 HN squamoustumors, 1/2 adenolung tumors. Low or no expression in normal tissues. SQL1 L970P 498531269 Highly overexpressed in 1/7 lung squamous tumors and 1/3 HNsquamous tumor. Low or no expression in normal tissues. SQL1 L981P 498651272 Over-expressed in 1/6 squamous lung and 1/3 HN squamous tumors. Lowor no expression in normal tissues. SQL1 L980P 49826 1279 Over-expressedin 3/7 squamous lung 1792 1807 tumors, 1/3 HN squamous tumors, 1/2 adenolung tumors. Low or no expression in normal tissues. SCL1 L973P 20631 117 Over-expressed in atypical carcinoid 1793 1808 METs andadenocarcinoma. Expression in several normal tissues. SCL1 L974P 20661 128 Over-expressed in primary small cell, 1794 1809 squamous andadenocarcinomas. Expression observed in several normal tissues. SCL1L996P 50430 1442 Over-expressed in 2/2 Primary Small 1795 1810 Cell, 6/6Small Cell Cell Lines, 1/1 Atypical Carc. METs, 1/1 Adeno, 1/1 Squamous.Very low or no expression in normal tissues. SCL3 L977P 26961  288Over-expressed in 1/2 Primary Small 1796 1811 Cell, 1/6 Small Cell-CellLine, and 1/1 Carcinoid Mets. Very low or no expression in normaltissues. SCL2 L978P 24928 1339 Over-expressed in 2/2 primary small 17971812 cell, 3/6 small cell-cell lines, 1/1 carcinoid mets., adeno andsquamous tumor pools; Low or no expression in normal tissues. SCL3/4L984P 50507 1446 Highly expressed in 1/2 primary small 1798 1813 celltumors and 4/6 small cell tumor cell lines. Low or no expression innormal tissues. SCL3/4 L580S 50536 1449 Over-expressed in select smallcell and squamous tumors. Some expression observed normal brain,bronchiol, soft palate and trachea. SCL3/4 L988P 50645 1531Over-expressed in 1/2 Primary Small 1799 1814 Cell, 1/2 Primary SmallCell, 6/6 Small Cell-Cell Lines, 1/1 Carcinoid Mets., Adeno & SquamousTumor pool. Expressed in some normal tissues (brain, adrenal gland,salivary gland, trachea, thymus). SCL3/4 L1423P 50625 1533Over-expressed in 1/2 primary small 1800 1815 cell , 5/6 small cell-celllines, 1/1 carcinoid mets. Also expressed in normal brain and pituitarygland. SCL3/4 L986P 50483 1490 Over-expressed in 1/2 primary small cell,516 small cell-cell lines, 1/1 carcinoid mets., adeno and squamous tumorpool. Expressed in normal brain, pituitary gland and spinal cord. SCL3/4L987P 50560 1527 Over-expression in 1/2 Primary Small 1801 1816-1818Cell, 6/6 Small Cell-Cell Lines, 1/1 Carcinoid Mets. Expression innormal pituitary and adrenal glands. SCL3/4 L1424P 50639 1547Over-expression in 1/2 Primary Small Cell and 1/1 Carcinoid Mets. Low orno expression in normal tissues. MLN1 L997P 26749  730 Over-expressionin 1/1 atypical carcinoid METs. No expression in normal tissues. MLN1L999P 26752  733 Over-expressed in 2/2 Primary Small Cell, 6/6 SmallCell Cell Lines, 1/1 Atypical Carc. METs. Expression in several normaltissues. MLN1 L1400P 26529  405 Over-expressed in 2/6 Small Cell CellLines and 2/2 Primary Small Cell. Moderate to low expression in severalnormal tissues. MLN1 L998P 27699  468 Over-expression in 1/1 Atypical1802 1819 Carcinoid METs. Low expression in normal tissues. LAP1 L1425P59303  949 Over-expressed in 4/7 squamous 1803 1820 tumors, 1/2adenocarcinoma tumors and in a pool of six small cell lung carcinomas.Moderate to high expression observed in normal brain, kidney andskeletal muscle. LAP1 L1426P 59314 1156 Highly overexpressed in one lung1804 1821 squamous tumor and one HN squamous tumor. Very low or noexpression observed in normal tissues. LAP1 L1427P 59298  921 Highlyover-expressed in 3/12 1805 1822 adenocarcinoma tumors. Very low or noexpression in normal tissues. LAP1 L1428P 59316 1180 Over-expressed in4/12 adenocarcinoma tumors and lower level expression in several otheradenocarcinoma tumors. Very low or no expression in normal tissues.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

SEQUENCE LISTING The patent contains a lengthy “Sequence Listing”section. A copy of the “Sequence Listing” is available in electronicform from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=06667154B1). An electroniccopy of the “Sequence Listing” will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. A method for determining the presence of a lungcancer in a patient, comprising the steps of: (a) obtaining a biologicalsample from the patient; (b) contacting the biological sample with anoligonucleotide or polynucleotide that hybridizes to a sequence setforth in SEQ ID NO:808, or a complement thereof complete, undermoderately stringent conditions; (c) detecting in the sample an amountof an expressed polynucleotide that hybridizes to the oligonucleotide;and (d) comparing the amount of polynucleotide that hybridizes to theoligonucleotide to a predetermined cut-off value, and therefromdetermining the presence or absence of the cancer in the patient.
 2. Amethod for monitoring the progression of a lung cancer in a patient,comprising the steps of: (a) contacting a biological sample obtainedfrom the patient with an oligonucleotide or polynucleotide thathybridizes to a sequence set forth in SEQ ID NO:808, or a complementthereof complete, under moderately stringent conditions; (b) detectingin the sample an amount of an expressed polynucleotide that hybridizesto the oligonucleotide; (c) repeating steps (a) and (b) using abiological sample obtained from the patient at a subsequent point intime; and (d) comparing the amount of expressed polynucleotide detectedin step (c) to the amount detected in step (b), and therefrom monitoringthe progression of the cancer in the patient.
 3. The method of claim 1,wherein the biological sample is selected from the group consisting of:sputum, blood, and bronchial lavage samples.
 4. The method of claim 2,wherein the biological sample is selected from the group consisting of:sputum, blood, and bronchial lavage samples.
 5. A method for determiningthe presence of a lung cancer or a head and neck cancer in a patient,comprising the steps of: (a) contacting a biological sample obtainedfrom a patient with at least two oligonticleotides under conditionswherein said oligonucleotides are effective for amplifying apolynucleotide sequence of SEQ ID NO: 808 in a reverse transcriptionpolymerase chain reaction; (b) detecting in the sample an amount ofpolynucleotide amplified in step (a); and (c) comparing the amount ofthe polynucleotide amplified in step (a) to a predetermined cut-offvalue, and therefrom determining the presence of a cancer in thepatient.